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Banana meriplants produced through tissue culture were tested
for suitability as material for screening for Fusarium wilt resistance.
The reaction of the meriplants after inoculation with known isolates of
Fusarium oxysporum f. sp. cubense was first compared with Musa
balbisiana seedlings. The disease reaction of the test meriplants was
evaluated after 60 days by scoring the above ground symptoms using
a rhizome discoloration index. The number of discolored rhizomes
from which F. oxysporum f. sp. cubense could be re-isolated was used as
the basis for percentage infection.

The results of the inoculation experiments established the
standards for evaluating the resistance of candidate clones when

suitability of banana meriplants fc
resistance has been verified with all tk
smaller plants. The standard F
the 6- to 8- leaf stage which were train
in cement beds and evaluated after
rhizome discoloration were advance
beds infested separately with identi
were evaluated after another 60 day:
the secondary test were multiplied
concrete culverts with sealed bottom
plants were subjected to a broad co
while their agronomic and commerce
in the field. A 4-fold increase (35%) i
were obtained from the population
secondary screening. Four clones v
in the secondary screening which w

screening for Panama disease
-herent advantages of handling
cedure used meriplants at
lanted into highly infested soil
days. The clones showing no
o the secondary test in cement
I virulent isolates. The plants
'lants that were not infected in
I tested simultaneously in the
id under field conditions. The
:tion of isolates in the culverts
potential were being observed
ie number of uninfected plants
* that were advanced to the
? identified as highly resistant
advanced to field testing.

60 Philipp. Phytopathol. 1995, Vol. 31(2):59-75
INTRODUCTION The development of a procedure fo
screening meriplants against a broao
Various methods of screening representation of fungal isolates causini
bananas for resistance to Fusarium wilt Fusarium wilt in the Philippines i
or Panama disease (PD) have been used. herewith described.
The most common procedure still being
used is to plant test varieties on
abandoned, heavily infested fields MATERIALS AND METHODS
(Vakili, 1965; Brandes, 1919). Where
adequate experimental areas are not All meriplants used in th
available, the test can also be done on experiments were supplied by a private
artificially infested plots (Stover, 1956), tissue culture laboratory. The youn;
concrete tanks or large clay pots (Stover meriplants were at 2- to 5-leaf stage (Fi
and Waite, 1960; Waite, 1977). Because 1). The greenhouse temperature range.
of the large plants being used, the test from as low as 70-76 'F to as high as 8C
plots necessarily involve large hectarage, 95 "F. Relative humidity ranged from 4(
enormous amounts of inoculum and 45% during the day and 100% during
from 8-36 months of field maintenance rainy days and usually after midnight.
work and observation. The screening
procedure is also subject to "escapes", Seeds of Musa balbisiana Colla wer
and it is very frustrating when calamities collected from mature bunches in a single
like typhoon, flood and other weather mat from the Bureau of Plant Industr
disturbances- occur during the Experiment Station at Bago-Oshirc
experiment (Osborne, 1958). The use of Davao City. The seeds were extract
meristem tissue culture plantlets manually, air dried for one week an
(meriplants) was explored for rapid stored in the refrigerator inside a plastic
increase of banana clones that have bag. The seeds were germinated b
commercial potential. The method soaking in clean tap water for eight day
also fits very well into a Fusarium before sowing in sterilized silty clay loar
wilt screening program because of the in seedboxes. The germinated seed
ease of producing disease-free plants were pricked and transferred int
in quantities which would be very another seedbox until 2-3 months. Thi
difficult and expensive with the standard was the age found most susceptible t
propagational methods previously F. oxysporum f. sp. cubense.
used for bananas. Because the
meriplants are small, handling of a large The inocula used were pathogen
number of asexually propagated plants isolates of F. oxysporuim f. sp. cubens
in a small area in relatively shorter test (FOC) maintained as air-dried myceli
periods would be convenient. It is also grown on PDA wrapped in sterilize
easy to experimentally manipulate the bond paper and stored in the refrigerate
plants during the early stages of tissue (Nash and Snyder, 1965). The culturE
culture to induce variability into. the were revived when needed, by planting
population. bits of the dried agar with myceli;

-owth on tresh FDA. Single-spore
iltures from 10-day old revived cultures
ere used to inoculate corn meal-sand
:MS) medium or sterilized dried
mnana leaf tissues for soil inoculation.
>ore suspensions from cultures grown
r 14 days on PDA were used during
ithogenicity tests. All evaluation of
verity were based on a disease severity
dex (DSI) for above ground disease
Imptoms, and rhizome discoloration
dex (RDI) for the degree of damage on
ie rhizomes. The DSI is based on
sease severity scale of 1 to 5, where: 1
no yellowing, plants are apparently
!althy; 2 = slight yellowing of the lower
waves; 3 = extensive yellowing of leaves
ith pronounced wilting; 4 = severe
!lowing with indications of recovery

Figure 1. Banana fruit trays used to
righl size for Panama disea.

UI

the unopened or newly opened leaves;
id 5 = plants are dead (Fig. 2). The
DI was based on a rhizome discolora-
)n rating of 0-3, where: 0 = the rhizomes
clean, no discoloration of vascular
indles can be observed; 1 = slight dis-
,loration involving less than 1/3 of the
izomes; 2 = discoloration of several
indles involving 1/3 to 1/2 of the rhi-
)mes; 3 = the whole stellar region of the
iizome shows discolored vascular
indles (Fig. 3). The discolored sliced
;sues of the rhizomes which showed a
ting of 1-3 were plated on Smith's
-NB medium (Nash and Snyder, 1962)
check for the growth of Fusariuim.
recentt infection was used in combi-
ition with DSI and RDI to evaluate the
distance of the test materials.

Figure 3. Rhi/mes of meriplants sliced open to show Ihe different degrees
of rhi/ome discoloration 60 days after inoculation; = rhi/ome
clean; = slight discoloration observed in less than 1/3 of the
rhiizome; 2 = rhi/ome discoloration affecting 1/2 of the rhi/omei
with tendendv lo spread 3 = the whole stellar region of the rhi/ome
I.- i

11

nto another seedbox containing
I soil. Pathogenicity of Cavendisl

were planted in 12-inch claypots and
allowed to recover for 3 weeks. Five
plants of each variety were inoculated
with CMS culture of the Cavendish and
Latundan isolates by mixing a handful
of infested sand-soil mixture (the
colonized CMS medium was mixed with
sterilized soil at a 3:1 ratio of sand-soil
mixture to CMS) with the top 1-inch
layer of the soil. The DSI, RDI and

clones were inoculated to determine if
any one or combinations of the
symptoms described below could be
used as an indicator of rhizome infection.
This is critical in the screening program
because normally the material is
sacrificed during evaluation. A non-
destructive alternative evaluation would
be desirable.

64
percent infection on the test plants were
recorded 60 days after inoculation.

Screening of treated meriplants for PD
resistance

Meriplants from the tissue culture
laboratory were transferred to the
nurseries when they were at the
2- to 4- leaf stage. The plantlets were
planted in sterilized silty loam
soil in community trays until they
had six to seven leaves. During the early
stages of screening, the meriplants
were transplanted in individual
4" x 5" black plastic bags and were
allowed to recover for 2 weeks. The
plants were inoculated using a mixture
of the Latundan and Cavendish isolates
growing in CMS medium.

Equal volumes of the colonized
medium were mixed and diluted with
three parts sand-soil mixture for every
volume of inoculum mixture. Fifty to 100
gm of this mixture was distributed
uniformly in a trench dug around the
base of each plant. This method proved
very tedious and it was difficult to follow
several plants of the same clone during
the process.

A better method developed later
used concrete soil tanks. These have
already been mentioned above. The
meriplants were transplanted by batches
into the concrete soil tanks containing
sterilized soil infested with 1:1 mixture
of the Cavendish and Latundan isolates
grown for 14 days in sterilized dried
banana leaf tissues. Fertilization was
done weekly by sprinkling with a
solution of Peters fertilizer
(manufactured by Robert B. Peters Inc.,

Philipp. Phytopathol. 1995, Vol. 31(2):59-75
Allentown, Pennsylvania, U.S.A.) used
after 2 weeks from transplanting at the
rate of 27 gm/gal. of water. All
evaluations were done after 60 days by
pulling out the individual plants and
scoring them for disease severity. The
RDI was taken by cutting the rhizomes
longitudinally into four sections and
scoring the degree of discoloration. The
discolored rhizome tissues from each
infected plant were surface disinfected in
5% chlorox for 1-2 minutes, blotted dry
in sterile tissue paper and planted on
Smiths PCNB medium (Nash and
Snyder, 1962). Evaluation was completed
after 5 days when the rhizome
discolorations were verified as caused by
F. oxysporum by the presence of fungal
growth from the infected tissues on
PCNB medium.

RESULTS AND DISCUSSION

Among the isolates of FOC in our
collection there were two dominant
types: the Cavendish isolates which
generally produce abundant, very fine,
moderately coarse to white to salmon-
colored mycelia and the Latundan
("Silk") isolates that generally produce
less dense mycelia and produce light to
deep purple pigment in the agar
medium. Both types have sporodochial
and non-sporodochial sub-groups with
varying tendencies for pionnotal growth.
Selection of a representative isolate from
each group was based on extensive
pathogenicity tests using M. balbisiana
seedlings. Typical results of a test for
pathogenicity are presented in Table 1.
Experience from field collections and
with greenhouse inoculated plants
showed that isolates obtained from

hilipp. Phytopathol. 1995, Vol. 31(2):59-75 65

ifected tissue above the corm and pseu- The 2- to 3-month old seedlings were
ostem were highly pathogenic. All sub- very susceptible to FOC. Both the
equent collection of samples for isola- Cavendish and Latundan isolates readily
on were obtained from these tissues. infected the M. balbisiana seedlings but
the Latundan isolates produced a slightly
ifluence of plant age on susceptibility higher infection than the Cavendish
f M. balbisiana seedlings isolate. The resistance of older seedldings
of M. balbisiana to FOC agreed with
The results showed that the number Vakili's (1965) results where he identified
F wilted plants increased during 7 to 21 three reactions to inoculation of
ays after inoculation (Table 2). Recovery M. balbisiana with FOC, namely: i)
om wilting of some plants and lower seedling and mature plant susceptible;
percentage of infection was shown by ii) seedling susceptible, but mature plant
!edlings which were 4-7 months old. resistant; and iii) seedling and. mature

'Based on number of plants infected as shown by wilting and yellowing typical of Fusarium wilt infectic
means with the same letters are not significantly different using DMRT at 5% level of significance; D.
= days after inoculation

plant resistant. Among the varieties he reduction in plant height and RDI f
tested, only M. balbisiana exhibited the M. balbisiana seedlings (Table 3). TI
seedling susceptible, but mature plant responses were not as good in the ca
resistant reaction. Waite (1977), however, of the Cavendish meriplants (Table ,
reported the occasional infection of The number of roots infected in tl
mature M. balbisiana growing in highly meriplants (1-2 roots/plant) did n
infested soil in concrete tanks. Two-to increase correspondingly to the numb
three-month old M. balbisiana seedlings of roots present during inoculation. TI
were consequently chosen as a suitable agreed with the observations of previo
susceptible check in the screening workers (Beckman et al., 1961; Risbet
procedure. 1957; Wardlaw, 1930) that only a very fe
roots need to be infected for success
Reaction of M. balbisiana seedlings colonization of the rhizomes. Young ar
and Cavendish meriplants to FOC vigorously growing roots were al!
inoculation considered by these authors to be mo
resistant than older roots. Successf
The results of three experiments infections were, however, common

ean Ulrterence in average height ot 1I In oculated and uninloculated plants ot approximately the same height during
iculation
ean difference in number of roots of inoculated and iruinoculated plants
'terage number of roots with rhizome discoloration and positive isolation of F. oxspormn f. sp. rcidinse using Smith's
NH medium

able 4. Reaction of meriplants of four Cavendish bananas to inoculation with
Fusariun ox/yspjorlti f. sp. c'ibcfns

tean difference in average height of 10 inoculated and uninoculated plants of approximately.the
me heights during inoculation, the negative sign means a decrease in measurement
lean difference in number of roots of inoculated and uninoculated plants, the negative sign means a
'crease in measurement
average lumber of roots with rhi/come discolor tion and positive isola ioin of 1. in-/lpr nii f sp iiIh'ns
uing Smith's I'CNh medium
L)l=rhi/oime discoloration index

68
and Hijo Giant Cavendish, two field
susceptible varieties, showed mor
infected roots than Roy Kerr an
Umalag. The medium to large size
meriplants were also observed to be th
most suitable size for inoculation. Small
plants were sensitive to die back due t
transplanting and to increased rhizome
infection by other saprophytic organism
(Table 5).

The results of additional preliminary
experiments showed that an inoculun
from chopped infected tissues (sizes o
less than 0.5 cm) from the field can giv
comparable disease severity ratings an
percent infection on M. balbisiana as front
CMS culture or spore suspension of th
fungus on PDA culture (Table 6). Often
however, the readings were complicate
by other organisms associated with fiel
infected tissues. It was found later tha
the use of sterilized dried banana lea
tissues collected from plants unspraye
with fungicides for leaf disease control
were easier to handle and thus, suitable
as a carrier of inoculum for soi
inoculation of FOC (Table 7). In addition
a 50:50 garden soil and washed river san
mixture was suitable as a pottin
medium for inoculation wor
(Table 8).

Large concrete tanks 1 m wide x 3
long and 1 m deep divided into there
chambers to contain 1 m3 of soil eac
were found ideal for screening work. Th
chambers can be conveniently used t
provide three replications per batch c
materials to be screened and the soil ca
be maintained at a high inoculum titr
under identical environmental condition
(Fig. 4).

CL -InL UL JPL Va LLU.IL L siL UamL.1 L.1
e concrete soil tanks was conducted using
d separate and mixed inoculum of tl
d Cavendish and Latundan isolates mixe
e with the top 2- to 3-inch layer of soil. Ti
ll fungus was allowed to colonize the sc
o for 5 days. A plastic cover was provide
e to produce warm and moist condition
.s favorable for growth and the establish
ment of the fungus in the soi
Two-month old seedlings ofM. balbisia?
y and meriplants at 6- to 8- leaf stage wei
n carefully pulled from the seedboxE
)f and were immediately transplanted ini
e the inoculated soil in the concrete tank
d The plants were watered only when tt
n soil was less than field capacity I
e provide variation in soil moistui
I, conditions known to favor Fusarium wi
d infection.
d
it The four experimental clones use
f in this test were selected froi
d preliminary inoculation tests usir
1, meriplants planted in individual plast
le bags. M. balbisiana seedlings and Gr<
il Michel and Hijo Giant Cavendis
i, meriplants were used as standards fi
d comparison.
g
k The results confirmed the suitability
of the concrete soil tanks for screenir
purposes (Table 9). Using the RDI alor
n over-estimated the pathogenicity of tl
*e isolates because F. oxysporum was n
h isolated from several rhizomes showir
ie discolorations. Successful isolation of t]
:o fungus from the discolored tissues w-
f therefore necessary to confirm tl
n susceptibility to a given isolate. Th
re became the standard procedure f,
is evaluation of all candidate materials th
NAYrarn cormonrl fnr PT) raciafAncp

lipp. rnytopatnol. 1995, vol. 31m2):7b6-88 69

ble 5. Pathogenicity of the Cavendish isolate of Fusarimn oxysponru f. sp. cuibensc
to meriplants of Umalag and Hijo Cavendish separated into different size
groups

Size of
inoculated Number of No. of roots
Variety plants roots' infected2 RDP

Twenty grams of corn meal-sand medium or chopped rhi/(me tissues coloni/ed by the tungus
Placed directly under the rhi/nome of the seedlings, the roots of which were cut 2 inches from the,
e before transolantinnz: The roots of the seedling, were trimmed to 2 inche-, from the base and

The information obtained in the
preliminary experiments was consoli-
dated. It showed that M. balbisiana
seedlings and the meriplants have
similar expressions of Fusarium wilt
infection. The yellowing and buckling
of the leaves can be easily counted, but
early expressions are often complicated
with transplanting shock and very warm

he primary and secondary screening
ease using banana meriplants

of the year. Thus, leaf yellowing was
limited in usefulness for early screening.
The reduction in plant height can also be
quantitatively measured but it would
take a relatively long period for this effect
to be expressed by the affected plants. It
would also require uninoculated check
plants for comparisons every time.

The most reliable indicator or early

Table 9. Reaction of meriplants of four
clones of Umalag inoculated witl
isolates using concrete tanks'

Cavendish is
No. of
plants
Variety inoculated DSI RI

M. ballhisinna 27 4.2

Gros Michel 27 1.8

LJmalag (B6) 27 1.3

Hijo Giant
Cavendish 28 1.0

E9-OA-2-5-7* 15 1.8

E9-(A-2-5-10* 15 1.2 ]

E9-OB-1-8-2* 9 1.0

E5-11d-3-4-6* 9 1.0

Average 1.7

Evaluation was done 60 days after transplanting i
separate and mixed cultures of the Cavendish and
equal proportion before incorporation with the to[
in successful isolation of F. c.v!ypolior f1. sp. coello'st
eiverity index, RDI = rhizome discoloration index.

'Clones of Umalag
seedlings as well as in mature plants was
:he discoloration of the vascular bundles
attacked by the fungus. Rhizome
discoloration was likewise expressed by
:he meriplants. It was observed,
however, that infections of the roots and
rotted rhizome tissues may involve other
organisms including saprophytic
. oxysponrun, hence it would require a test
)f pathogenicity to confirm the cause of
Discoloration. It was also found that
solution of F. oxysporum from advancing
nargins of discoloration usually yielded
*^ t-l~n o ;-i ~ a- j

74
Both isolates produced a hig
percentage of infection on all the clone
inoculated (Table 10). Disease severit
and rhizome discoloration, however
varied among the clones and between th
two isolates used. Higher DSI and RE
and percent infection were produced b
PD 68 compared to PD 59. The procedure
appeared sensitive in differentiatin
degrees of susceptibility among clone
coming from a single accession. It ca
be seen from these data that clones B6-]
B6-7 and B6-17 can be advanced t
additional tests and probably increase'
as a possible resistant population.

There was an almost 5-fold increase
in the number of uninfected plants fror
the selections advanced to the secondary
screening (Table 11). The screenin
procedure was able to select clones wit
higher resistance to the disease. Thes
lines can be further multiplied and r(
tested against a broad selection of isolate
representing the fungal population
existing in the Davao area. Promisin
clones can be identified and planted i
infected sites in the commercial

A collection of banana varieties was established adjacent to two
commercial banana plantations in Davao del Norte and Bago-Oshiro,
Bureau of Plant Industry Experiment Station, Mintal, Davao City. These
collections were tested for Panama disease resistance. Priority was
given to varieties with commercial potentials.

Preliminary observations of the varietal reaction to virulent isolates
of Fusarium oxysporum f. sp. cubense were conducted using meriplants
planted in 12-inch clay pots in the greenhouse. Evaluation were based
on the disease severity index and the rhizome discoloration index taken
60 days after inoculation. Varieties exhibiting resistance were planted
around newly eradicated infected mats in the field. The varieties were
considered susceptible when they become infected. Those.that
remained uninfected were multiplied and planted in additional sites.

The experiment identified 41 highly susceptible varieties. Among
the AAAs, Ipod and a Roy Kerr accession have shown resistance to
Panama disease. The ABBs were observed resistant under field
conditions. Of the ABBs, onlv Latundan Tsina had shown resistance

iiupp. rnytopamoi. itvy, vol. slw.:7-o- 77
INTRODUCTION f. sp. cl'?cnnsc. Vakili (1965) showed
differential wilt resistance to race 1 and 2
Fusarium wilt or Panama disease in seedlings of M. acuininata subspp.
D) as it is often called, is still one of the erra ns, banksii, and iiicrocaria.
ast dreaded disease of bananas. Segregation of hybrid seedlings of three
nmonds (1965) included it with the Musa sp. x Lidi crosses indicated a single
If dozen most catastrophic diseases, dominant factor for resistance to race 1
inparable in importance with wheat in Lidi. He also observed that M.
;t and potato blight. The disease was balbisiana was the only species tested
gely responsible for the destruction of which displayed seedling susceptibility
ousands of hectares of bananas in and mature plant resistance to Fusarium
)pical America where the susceptible wilt.
ne Gros Michel was grown. PD was
o the reason for the change from Gros Stover (-1957) studied the
chel to the PD-resistant Cavendish pathogenicity of the two types of F.
oup. The fear of PD becoming a oxyIsporiui f. sp. cubecusc and Stover and
)blem on Cavendish developed, when Waite (1960) describe race 1 as highly
iwan started loosing approximately pathogenic on Gros Michel, but not on
00 ha of Cavendish because of this Bloggue, whereas, race 2 was highly
-ease (Su et al., 1977). pathogenic on Bloggue but slightly on
Gros Michel. Waite (1977) used Gros
Lee and Serrano (1920) were the first Michel and Bloggue as indicator plants
report the presence of PD in the to identify races 1 and 2 of F. oxyspornim.
ilippines which was attacking He identified the isolate from infected
tundan at Los Bafios and Calamba, Latundan from the Philippines as race 2
guna and municipalities of Batangas. which confirm the finding of Vakili
eir inoculation work on Miusa textilis (1965). In field inoculations with 57
ing isolates from Latundan gave banana species and hybrids of adult
;ative results. Because of the danger plants, varying degrees of susceptibility
sed by the disease on the flourishing or resistance were noted among AA,
ica industry during the period, most AAA, ABB, and BB varieties to race 1.
the works dealt with investigations According to Waite (1977) Stover tested
citing banana wilt with abaca wilt and a Fuisarium isolate from diseased
ting resistance of abaca varieties Latundan from Taiwan and identified it
iinst the banana wilt organism (Lee as race 1. Stover believed that the
d Serrano, 1920; Teodoro, 1925; Philippine Latundan was apparently
oncio, 1930; Palo and Calinisan, 1939; different from Latundan or Silk Fig of
still and Celino, 1940). Taiwan. The Philippine Latundan has
been equated with Pisang rasthlli (AAB)
A similar study was undertaken by which is synonymous with Silk
nali et al. (1956) when they reported (Simmonds, 1965; Valmayor, 1980). Silk
t seedlings of Pacol (a clone of Misa is also equated with Manzano in
b'isiana as reported by Allen, 1965), Venezuela (Waite, 1977), but then,
re susceptible to Fiusariiiiii oxlspolrin Manzano in Venezuela is different from

the Philippine and Taiwan Latundan. have pigmentation (light to deep purple)
Otherwise, a different race of the darker than the isolates from Cavendish
pathogen was involved. (creamy white to salmon or flesh
colored).
Our observations of the Latundan
(AAB) in the Davao area showed this The Philippine banana export
clone to be highly susceptible. It is very industry is based on clones belonging to
common to see infected Latundan plants the Cavendish group such as Hijo Giant
growing among very healthy Saba (ABB) Cavendish, Robusta, Valery, Umalag and
in backyard plantings. Attempts to grow Grand Nain in varying number of
Latundan in commercial orchards in hectarage planted. The current total area
three widely separated areas 'by planted by all the banana growers is
independent banana growers showed 75- approximately 30,000 ha located mostly
80% of the plants infected with Fusarium in Davao del Norte, Davao City, Davao
wilt in less than two years. del Sur and South Cotabato.

Recent works on varietal resistance Panama disease was first confirmed
to PD agree with the extreme in the commercial plantation in 1974. The
susceptibility of Gros Michel to race 1. increasing number of cases since then
Clones I.C. 1 and I.C. 2 (from a cross have emphasized the potential problem.
between Gros Michel and M. acuninata), A search for new resistance to the disease
Valery, Lacatan, Manzano, Giant became a necessity.

and Ioyo are all resistant to ru (reaKen,
1971). The change from Gros Michel tc
"Lacatan" in Jamaica and other Tropical
American countries was based on the
resistance of "Lacatan" (Simmonds,
1965). When Giant Cavendish became
infected in localized areas in Taiwan,
Stover (as cited by Waite, 1977) stated that
the Fusarium from Latundan in Taiwan
may be different from the one attacking
Cavendish. Su et al. (1977) reported that
the race attacking Cavendish in Taiwan
is a different race and identified it as race
4 of F. oxysporum f. sp. cubense. Waite
(1963) reported F. oxysporum f. sp. cubensc
race 3 on Heliconia spp. Our fungal
isolations from Fusarium wilt infected
bananas in the greater Davao area and
an area in Bukidnon showed culturally
distinct isolates from Latundan and from
Cavendish. The isolates from Latundan

-i Danana variety collection was
initiated and screening for resistance to
the locally existing isolates of the causal
organism was conducted in the
greenhouse and infected sites in the field.
This report covers the greenhouse and
field screening of banana varieties for
resistance to Panama disease.

MATERIALS AND METHODS

An extensive collection of wild and
cultivated clones of banana was
established by Philippine Packing
Corporation in 1978. Additional
accessions were included from collection
trips to Mindoro, Surigao, Misamis,
Davao del Norte and from the Banana
Gene Bank of the College of Agriculture,
UPLB and the Bureau of Plant Industry

Inoculation conducted in the
greenhouse were done using plantlets
produced by meristem tissue culture
technique (meriplants) from a private
laboratory. The selection of the accessions
for multiplication was based on the
central objective of identifying early in
the screening program, PD-resistant
clones that could be used in a
conventional breeding program, or
varieties that already possessed the
useful agronomic characters of
Cavendish that could be used
immediately to replant PD infected areas.
Meriplant production was therefore
concentrated on the AA and AAA clones.
The AAB and ABB clones were mostly
used as standards for comparison of
resistance.

The meriplants were brought to the
nurseries when they were at the 2- to 4-
leaf stage. The plantlets were planted in
sterilized silty loam soil for 4-6 weeks
after which they were transferred to
10-inch clay pots filled with sterilized
1:1 mixture of washed river sand and
silty clay loam soil. Ten plants per
accession were planted individually in
the pots and allowed to recover for 2-3
weeks u.der partial shade outside of the
greenhouse. Inoculation was done by
mixing 20-30 grams of corn meal-sand
culture of the inoculum in a. shallow
trench about 2-3 inches around the base

or eacn plant. ne plants were tertlized
weekly by sprinkling with Peters
fertilizer (20-20-20 NPK at a rate of.27
gm/gal water (manufactured by Robert
B. Peters C., Inc., Allentown, PA, USA).
Malathion EC (57% Malathion at the rate
of 20 ml/1) was sprayed once every
month to control insect pests and mites.

Field screening

Suckers of candidates materials for
field testing were dug from the field
nurseries and brought to the commercial
plantations on an irregular schedule
depending on the availability of open
space in the infected field sites. This
condition limited the number of plants
that could be tested at any given time and
the result likewise did not allow
comparison of degrees of resistance
among the test materials. Four suckers
of .different test clones were planted
equidistantly around every eradicated
infected mat in the test site. Each plant
in the site was provided with a stake
indicating the date of planting and name
of the clone. The length of exposure in
the test site can be computed when the
entry becomes infected based on the date
indicated on the stakes. The usaal
number of mats per test clone was five.
This number was increased to 15 for
those entries with no report of infection
after 24 months, or results in the
laboratory inoculations indicated a high
level of resistance. If a variety had
developed into infected mat, that variety
was immediately taken out of the site to
make space for additional materials.
The varieties in the test sites were given
the standard cultural practices for care
and maintenance.

RESULTS AND DISCUSSION

The clones which were collected and
maintained in the banana collection
nurseries are listed in Table 1. There were
70 accessions of 121 clones partly
classifiedd into their respective clonal
;roups. There are some duplications in
erms of synonymy as verified in the list
)f Allen (1965) and Valmayor (1980). The
lumber of local names and dialects that
ire associated with the name of a clone
indicate the distribution and the extent
)f the usefulness of the clone in this
country. The name pertaining to the AAs
ire used interchangeably probably
because of their generally similar stature

Philipp. Phytopathol. 1995, Vol. 31(2):76-88
and Saba (ABB) showed differences in
their reaction to four isolates of
F. oxysporum f. sp. cubense (Table 2). Gros
Michel was more susceptible to the
Latundan isolate than the isolates from
Cavendish. The reaction of Saba varied
from resistance to immune when
inoculated with the Cavendish isolate
and only moderately resistant to the
Latundan isolate. It was therefore
suspected that the Cavendish and
Latundan isolates have different levels of
pathogenicity. Subsequently, the
greenhouse inoculations were done
using both the Cavendish and Latundan

'able 1. Banana varieties used in
screening for resistance to
Panama disease

imaIii 1uii, ri- 111
Clonal Group Clonal Name' Moco (Saba type, ABE
Mocol (Isarog, BB)
Ternate Pepita
Tindok (3) Pusdan
Berocsoc (AAB)
ABB Awak 'Number in parenthesis are the number of accessior
Benayoy colUlcted of each clones. Those which wetr not indicate
Biinenito have only one. Duplicate clones are not necessarily
identical since they have been collected in differed
Cadiznon locations.
Calapagnan (2)

Cardaba growth rate. Vietnam is another
Costa Cavendish accession of the Grand Nair

Matavia

Penipita
Profeta
Saba
Saba Local
Silvery Matavia

BB Botohan
M. balbisiana (3)
Paquel (2)
Puntacon

AAAA Bodies Altafort

ABBB Tiparot

Unclassified
Ploidies Acc 49
Acc 147
Acc 148
ienanga r
Botolan (AA)

Dohoy
Duoy
Good (AAA)
Kahuslot
Kalunsmlg

Kanaa
Kinagan
Kinatsila
Konikit

reaction in this test. Roy Kerr apparently
is a variety which exhibits field
resistance. The tetraploid Bodies Altafoi
(AAAA) was found very susceptible t
both isolates. Only Latundan Tsin
showed resistant reaction to the isolate
tested among the AAB. Two accession
of Saba showed different reactions t
F. oxysporuin f. sp. cubense inoculatior
One was moderately susceptible to bot
isolates while the other was moderate
resistant to the Latundan isolate an
resistant to the Cavendish isolate. Thi
reaction does not agree with the observe
resistance of Saba under field condition!
It also point out the need to retain th
individual identity of duplicate
accessions. Accessions from differer
geographic areas may differ from on
another in disease reactions.

Field testing

Because of the absence of artificial
inoculation, information on the generic
susceptibility of the clone being teste,
can be obtained only if infection occur
Uninfected clones will require planting

11Vt LUILc U i LZO IV U IlCl

Clone Clone Name Isolate Used Disease Rating2 Number of P
Group for Inoculation' RDI Reaction Infected/Inocul

'Five isolates were selected from the collection; PD 28 was an isolate from Green Bongolan infected in one o. tl
sites in Lapanday; PD 33 from infected Hijo Giant Cavendish at Evergreen; PD 59 from Tumok in one of the tes
at Lapanday; and PD 68 from Latundan adjacent to the Cavendish plantations at Farmingtown.

in more sites with extended periods of The test sites were all inside tl
observations. Simmonds (1965) listed commercial plantations and in sow
the following factors that affect the cases Cavendishwas eradicatedbecau,
occurrence and course of Panama disease of PD. The reaction of each clone w,
in the field: 1) the clones grown is the compared with the greenhous
most important single factor which can inoculation results using the Cavendis
decisively influence the course of isolate. Very good agreement in tl
infection, 2) age of the plant, 3) weather greenhouse and field tests for AA, AA
influencing the course of infection, and AAAA (Bodles Altafort) clones wei
4) general condition of the roots of the obtained (Table 3 and 4). The ABB groul
rnlfner-c r\ rlhryl cnri I-r1 f fhi ccil c~ hdhn l whih wcr.- pvtrcm0hr C1,CCPuI-hlP l

and 8) the amount of inoculum available
Ideally, the test materials should b
planted so that each plant would I
subjected to the same inoculum level
a given period of time. From these field
tests, we identified the varieties listed i
Table 4 as susceptible to Panama disease
These varieties became infected while
growing as replants on PD infected site

5 years in the test sites. This may reflect
the interaction of plant age and disease
susceptibility. The field resistance of th
ABB group is also supported by th
absence of PD infected Saba or Cardab
even if they are interplanted among;
heavily infected Latundan. There wer
two documented cases of Panam
disease infection on Saba in 1974 an,

-1

iilipp. Phytopathol. 1995, Vol. 31(2):76-88
)75 by J.P. Silva (personal
)mmunication). However, the same
rmptoms have not been observed on
Iis variety after these reported cases.
ie ABB are apparently very resistant
under field conditions but very
isceptible as meriplants.

Among the AAA which were
>served resistant in the greenhouse,
iay Baguio and Vietnam were infected
hen planted in three test sites. It was
)ted also that the two accessions of Roy
err have shown differences in their
action to Panama disease in the test
tes. Roy Kerr (Acc. 69), an accession
am Marsman, has shown several cases
' infected plants but Roy Kerr (Acc. 4)
om Hijo plantation has not shown
ilting although in one instance very
united infection in the rhizome of one
ant was seen. The procedure used in
ie testing was limited by the restriction
i private companies to intentionally
crease inoculum level in the soil
!cause of the accountability to clean the
fected soil after the test is completed.
s a consequence very few test plants
came infected in the field test
)mpared to the greenhouse tests (see
ible 5). Such accountability problem
in be solved if a banana research station
in be established.

stitute of Plant Breeding, UPLB, and
thology, University of the Philippines

stance, Rhizoctonia solani, sorghum,

CT

:rosses involving a resistant
ety, UPL Sg5 indicated that

however, appears to play a bigger role.

INTRODUCTION diseases, and weeds all constantly exert
a harsh selection pressure (Doggett,
Sorghum is cultivated widely 1970).
throughout the tropical, subtropical, and
emperate regions within the latitudes 45 In the Philippines, sorghum
QJ and 45 S. It is grown largely in Africa, production is quite small. The grain is
sia, the Americas, Australia, and in primarily used as animal feed. Diseases
egions that are generally too dry for are considered as one of the limiting
consistent, reliable maize production. In factors in production. Of the 13 diseases
northh America and Australia, sorghum reported to occur locally (Karganilla and
3 grown commercially in large farms and Elazegui, 1973), Rhizoctonia sheath
he grain is used principally for animal blight is considered one of the most
eed. In less-developed tropical countries, important. This disease, characterized by
orghum is produced predominantly on the presence of irregular blotches on the
>easant farms where yields are low and leaf sheath showing typical banded
;rains are used mainly as human food. pattern, is caused by the soil-borne
pathogen, Rhizoctonia solani Kuhn which
Sorghums in the tropics have belongs to a group with high plasticity
volved in hostile environment, where and consequently with a wide host range
inrolirnhl rn;nfnll -nnr cnile nocp- nnd wnrldwidp dintribiitinn The

90

corn, wheat, and many other crops
(Parmeter and Whitney, 1970). The Reciprocal crosses involving tl
disease, which normally starts from the resistant line, CS 621 and the susceptib
basal leafsheaths and spread upwards variety, UPL Sg5 were made an
reaching as high as the panicle and subsequently advanced to F,.
causing wilting of the affected areas, can populations were crossed to both paren
be a serious problem in areas of light rains to obtain BCIA and BCIB, backcrosse
with prolonged duration. The pathogen to CS 621 and to UPL Sg5, respective]
survives between cropping seasons in and at the same time advanced to
crop debris in the form of mycelia and generation. The parental lines, F,, F, ar
in the soil as sclerotia. backcross populations were planted
a randomized complete block desi
Yield losses in sorghum due to with four replications where hills we
Rhizoctonia sheath blight range from 10 cm apart and rows 60 cm apart in
35-42% (Pascual and Raymundo, 1988). nursery. The plants were inoculated wil
The.use of resistant varieties appears to R. solani at 30 days after emergence t
be the most economical and effective the leaf sheath inoculation methc
method of controlling the disease. Some (Pascual and Raymundo, 1988). 1
lines of sorghum have been identified maturity, plant height and lesion size ,
as moderately resistant to Rhizoctonia individual plants were measures
1 t 1 1" ,IIT 'l'l' ., dnnJ' 1T .

1992). Earlier, it was used, with a sisti
line, CS-622, in a study of tl
epidemiological parameters of resistance
to R. solani (Pascual and Raymundi
1989). Relative to the susceptible cultiva
UPL Sg5, resistance reduced the rate (
disease development by 40.3%. Th
resistance components involved i
reducing the rate were lower relative
lesion height and fewer number c
sclerotial bodies. Abetter understanding
of the genetics of host-pathoge
interaction is needed to make disease
resistance a more effective, efficient an
lasting control measure. Unfortunately'
information regarding gene complex
conditioning resistance to Rhizoctoni
sheath blight in sorghum is not available
This paper reports on the mode c
inheritance of resistance in the sorghur

to the length of stem covered with sheal
blight infection.

Frequency distributions of tb
different populations were determine,
Generation means on relative lesio
height data were analyzed according I
the procedure of Gambie (1961a, 1961b

RESULTS AND DISCUSSION

The environment after inoculation,
30 days after plant eIne-gence we
favorable enough for sheath bligl
development as to allow discrimination
between susceptible and resistar
genotypes (Table 1). Differences among
mean lesion heights of parents an
different progenies were observed. Th

sistant parent, CS 621, at 21.17 was susceptibility in the F,. Additive
significantly different from that of the variance, likewise is highly significant.
isceptible parent, UPL Sg5, which was
.58 and the other populations. The F2 CS 621 was the first sorghum line
ean relative lesion height, 34.42, was found to have significant degree of
wer than that of the F1, 49.92. resistance to R. solani (Pascual and
Raymundo, 1989, 1992). Symptom
Frequency distributions for relative expression was in the form of reduction
sion height of different generations of in the severity of disease and slowing
e cross between CS 621 and UPL Sg5 down of the infection rate (Pascual and
e shown in Fig. 1. The F, distribution Raymundo, 1989). Evidences of
skewed towards the susceptible parent hypersensitive types similar to those
iggesting dominant gene action for found in rust (Hooker, 1967) were not
sceptibility. This tendency was also seen. The quantitative nature of the
,parent in the backcross populations genetics of resistance exhibited by CS 621
here the level of disease severity was is similar to that observed in many types
gher than that of the resistant parent. of diseases in cereal crops. For instance,
in a quantitative resistance analysis
The additive-dominance model involving Cercospora zeae-maydis in corn,
,pears able to explain the expression of additive genetic variance was shown to
lantitative resistance to R. solani in CS be the major source of genetic variation
1 (Table 2). Variances for additive, (Thompson et al., 1987). Raymundo
iminance, and additive x additive gene (1991) reported that in a Design I
fects were positive while that for mating system utilized to estimate
[ditive x dominance effect was genetic parameters for resistance to
!gative. The estimates showed a Puccinia polysora in corn, additive genetic
bstantial and significant amount of variance is substantially higher than'non-
iminance genetic variance which can additive variance. Estimates of additive
indicative of the expression of genetic variances were usually larger

92

100

80-

60

40

20

o 0
0-10 11-20 21-30

(U
-10
LL

8-

iF1
6-

4

2

0
0-10 11-20 21-30
Cla
Relative IE

Figure 1. Frequency distribution of ,
~

*F2
6 BC1A
*BC1B

31-40 41-60 61-80

31-40 41-60 61-80
ses
ion height

different generations of the cross

able 2. Estimates of genetic variance for re
generations of crosses between C

Respectively, Science Research Assistc
otection, Visayas State College of Agricult

Keywords: inoculum density, Meloidogi
rnatode, upland rice

ABSTR

A study was conducted to d(
inoculum densities of the root-knot ni
on upland rice cv UPLRi 5 and to evw
cultivars/lines to the nematode.

Meloidogyne gramninicola at vai
reduced the number of tillers, planI
weights of rice cultivar (UPLRi 5) cor
Based on grain weight, yield reducti
number of galls and eggs produced v
the treatments.

Among the 51 cultivars/lines e
(IR 62761-20, IR 60080-48 and IR!
resistant to the nematode based on t

INTRODUCTION

Rice (Oryza sativa L.) is considered
re of the most important cereal crops.
constitutes the staple food for most
developed and developing countries.
he 1992 world population is about
3 billion, and is estimated to increase
almost 100 million Deoole each year.

By 2020, the world must produce
350 million more tons of rice to meet the
demand of the increasing population
(IRRI, 1993). At present, however, the
land area devoted for lowland rice
production is getting smaller.
Urbanization had converted several
hectares of irrigated lands into
subdivisions. Therefore, it is necessary

mac production snouia De mtenslnea m
the remaining irrigated lands and in the
upland areas. In Asia, Africa, Latin
America, and the Carribean, about 19
million hectares of rice are grown in the
upland areas.

Upland rice production in the
Philippines is beset with pest
problems, including nematodes. Several
genera of nematodes were found t6 infest
upland rice and about six of these have
major significance as being rice pests
causing yield losses in an economic
proportion (Villanueva and Prot, 1992;
Soriano and Prot, 1992). Among the
nematodes associated with rice, the root-
knot nematodes have been considered
one -of the most destructive in the
Philippines.

Rootknot diseases of rice is caused
by several Mcloidogyne species.
Meloidogymn graininicola, which was
originally found on Echinochloa colony
Link., was discovered to infest rice in
Laos, India, Thailand, Japan, and South
Africa (Golden and Birchfield, 1968).
Van der Linde (1956) also reported from
South Africa that M. javanica (Treub)
Chitwood, M. arinaria (Neal) Chitwood,
M. incognita var. acrita Chitwood,
penetrate and reproduce in rice.

In the Philippines, root-knot
diseases of rice was reportedly caused
by M. incognita and M. graininicola (Fofie
and Raymundo, 1979). M. graininicola is
the most important nematode of rice
because they infect both lowland
and upland rice. It is widely distributed
and found in about 40% of the 18
provinces surveyed (Soriano and Prot,
1992).

environmentally unsafe. The use (
resistant varieties, therefore, offei
a good alternative. To date, the availab
information on resistant varieties an
their role in nematode control is still vei
limited. For example in India, Israel i
al. (1964) reported that rice cultivars diff(
in their susceptibility to M. incognita va
acrita. Ch. 47 had an average of only 0
galls per plant while HR-19 had 53
galls, others- were intermediate i
susceptibility. Roy (1973) tested 1C
cultivars and found the varieties Gare
and Dumal resistant while the others ha
varying degrees of susceptibility. Yik an
Birchfield (1979) reported that among 2
cultivars they tested, LA 110 and Bonni
were resistant, nine were moderate
resistant, 13 were susceptible, and tw
were very susceptible. Villanueva an
Prot (1992) tested nine upland ric
cultivars for resistance to M. graininico
and all the cultivars were foun
susceptible to the nematode. This stud
was therefore conducted to determine tt
pathogenicity of M. graminicola o
upland rice (UPLRi 5) and evaluate tl
reactions of upland rice cultivars/line
to the nematode.

MATERIALS AND METHODS

Experiment 1. Effect of Differer
Inoculum Densities

Field soil was sterilized at 60"(
for 2 hr, allowed to cool and transferred
to 30-cm diameter clay pots.

Complete fertilizer (14-14-14 NPK
was applied in each pot as basal fertilize

Philipp. Phytopathol. 1995, Vol. 31(2):95-102

at the rate of 50 g/pot. The fertilizer was
covered with soil about 2 cm deep and
seeds of upland lice (UPLRi 5) were sown
in each pot. At milk stage, rice panicles
were bagged to protect them from
infestation by rice bugs and birds.

Eggs of M. grainiicola were
extracted from roots of infested rice
(UPLRi 5) plants using the methods of
Hussey and Barker (1973).

Plants were inoculated by pouring
the egg suspension into the holes made
near the base of the plant using a pipette.
After inoculation, the holes were covered
with soil to keep the nematode eggs from
desiccation.

Potted plants were arranged in a
randomized complete block design
(RCBD) with the following treatments:
0, 100, 500, 1000, 5000, and 10000 eggs/
pot. Each treatment was replicated five
times. The Duncan's Multiple Range Test
(DMRT) was used to test means
differences.

The plants were harvested at
physiological maturity and the following
data were gathered: number of tillers,
plant height, herbage weight, root
weight, grain weight, number of galls,
and number of eggs.

Experiment 2. Reaction of Upland Rice
Line/Cultivars

Field soils were sterilized as
described in Experiment 1 and later
transferred into 15-cm diameter clay
pots. The pots were arranged in a

complete randomized design (CRD) with
five replications. Dried seeds of different
cultivars and lines of upland rice
obtained from the Department of
Agronomy, Department of Plant
Breeding and Agricultural Botany, and
Farm and Resource Management
Institute (FARMI) of the Visayas
State College of Agriculture (ViSCA)
were sown into clay pots with sterilized
soil.

A total of 51 cultivars/lines of
upland rice was tested for their
reactions to the root-knot. The upland
rice cultivar UPLRi 5 was used as
susceptible check. DMRT was used to
test mean differences.

Sufficient moisture was maintained
by watering each plant whenever
necessary. At four-true leaf stage, the
test plants were thinned to one plant per
pot.

Preparation of inoculum and
inoculation were essentially the same
as in Experiment 1. M. graininicola
was inoculated at the rate of 1000 eggs/
pot.

Test plants were harvested 50 days
after inoculation. Galls from the
cleaned roots were counted and the eggs
were extracted and counted using the
method of Hussey and Barker (1973).
Resistance rating was based on the
gall index of Taylor and Sasser (1978)
as follows: 0-1.9 = Resistant (R), 2.0-2.9 =
Moderately Resistant (MR); 3.0-3.9 =
Moderately Susceptible (MS); 4.0-5.0 =
Susceptible (S).

98
RESULTS AND DISCUSSION

Experiment 1. Effect of Different
Inoculum Densities

The number of tillers, plant height,
weight of herbage and roots were
comparable in plants inoculated with- 100
eggs and the uninoculated control plants.
However, significant differences of the
above parameters were observed
between the uninoculated control plants
and those inoculated with 500, 1000,
5000, and 10000 eggs, except in plant
height (Table 1). Generally, as nematode
inoculum density was increased, the
values of the different plant growth
parameters decreased with maximum
reduction in plants inoculated with 10000
eggs.

Based on the grain yield. percentage
reduction of 31.75% was observed in
plants inoculated with 10000 eggs, while
plants inoculated with 100 eggs had 9.5%
yield reduction.

Significant differences on the
number of galls and eggs were observed
among the various treatments (Table 2).
The number of galls and eggs produced
were directly correlated with initial
inoculum density. As the inoculum
density was increased, the number of
galls and eggs produced in the rice plants
also increased.

Table 1. The effects of varying inoculum densities of Mcloidogyni graniniicola on the
number of tillers, plant height, weight of herbage, roots and grains of upland
rice cv UPLRi 5

CV(%) 13.31 4.67 7.38 17.21 8.20
'Percentage yield reduction was based on weight of grains in the control, in a column, means with the
same letters are not significantly different at 1". level of significance with DMRT

[able 2. Effect of initial inoculum densities <
of galls and eggs produced in up

Inoculum Number
Density per ti

Jninoculated control 01

100 39.!

500 55.(

1000 73.,

5000 127.

10000 144.1

CV(%) 10o.

In a column, means with the same letter are not si

The overall result of the experiment
owed that M. graininicola could
mage upland rice (UPLRi 5) at various
nsities as shown by the reduction of
e different growth parameters and
ain yield. The effect of nematode
!ding could have altered the normal
lysiological functioning of the plants.
ills that were produced could have
ered the synthesis and metabolism of
rbohydrate, amino acids, proteins,
Dwth regulators and others in the
ants (Owens and Specht, 1966;
glierchio and Yu, 1965). Although
ese physiological changes were not
assured they could have contributed
the low yield of rice especially at high
)culum density. Yield reduction was
o. reported in sweet potato, ampalaya,
d cassava inoculated with different

99

f M'loidogyne graiiniicola on the number
and rice cv UPLRi 5 at harvest time'

The number of galls and eggs
produced by the nematodes varied
significantly among cultivars/lines.
Among the 51 cultivars/lines evaluated
for their resistance to M. gramiinicola, 3
lines (IR 62761-20, IRAT 610, and IR
60080-48) were moderately resistant
(Table 3). These cultivars had the lowest
mean counts of galls and eggs which
ranged from 8.2 and 2325.0 to 8.8 and
2751.6, respectively. On the other hand,
BPI Ri 6, Binagakay, and Panatikoy were
the most susceptible cultivars.

1UU

Table 3. Number of galls and eggs prod
oculation with 1000 eggs of Melt

The low rate of nematode
reproduction in the cultivars considered
moderately resistant could be attributed
to the genetic, physiological, and
biochemical factors within the cultivars/
lines. These could limit the entrance of
nematode into the roots, thus, reducing
the of nematode penetration and
establishment within the root (Rohde,
1965). It is also possible that chemicals
(e.g. phenolics, phytoalexin) within the
host may affect the fecundity of the
female nematodes, thus, reducing their
rate of reproduction (Giebel, 1974; Veech,
1978).

lent and Professor of the Department of
Agriculture, (ViSCA), Baybay, Leyte,

talensis, mango stem-end rot, Trichoderina

WCT

inhibited mycelial growth and
f Diplodia iatalenisis in agar culture

In vivo efficacy test of T. viridc at 10" conidia/ml resulted in
significant reduction in disease incidence from 100 to 20% translating
into 80% level of protection of mango fruits. Treatment of
T. viridc at 10" conidia/ml provided 67.9 and 63.4% biocontrol efficacy
based on diseased fruit surface area and fruit volume rotten,
respectively. Such efficacy levels were comparable to that provided by
benomyl.

T. viridc completely protected mango fruits from being infected
when inoculum levels of D. natalcnsis was 15,000 conidia/ml or less.
The levels of biocontrol declined from 95-35% when pathogen inoculum
increased from 20,000-50,000 conidia/ml.

Simultaneous application of T. viridc and of D. natalensis caused
the lowest disease incidence and severity of stem-end rot. Delaying
T. viride application after inoculation of D. natalensis resulted in lower
disease control than before inoculation of the pathogen. T. viride alone
did not cause any observable lesion on treated mango fruits.

INTRODUCTION

Mango (Mangifera indica Linn.) is
regarded as high-valued fruit crop both
in local and export markets. Supporting
some 2.5 million farmers in the
Philippines, mango ranks third among
the most important fruit crops in terms
of production area, volume and value
(PCARRD, 1995). Its attractive
appearance, luscious flavor and aroma
delight both local and foreign consumers
and in 1993 exports from dried and other
processed mango products reached
10,813 tons valued at US$15,771.98
(PCARRD, 1995).

Philipp. Phytopathol. 1995, Vol. 31(2):103-116
Moreover, the longer the delay of hot
water treatment, the lower the level of
control achieved (Dodd et al., 1991). With
chemical control, Tongumpai et al. (1980)
found that benomyl at 250 ppm was
effective when applied within 2-24 hr as
post-harvest treatment. Bay-Peterson
(1988) reported that the combination of
benomyl application and hot water
treatment is a better control tactic against
the fungus.

In recent years, the observed adverse
effects of chemical pesticide residues on
human health and the development of

ror more innovative ana ecologically
Together with poor handling during sound control measures such as the
transport, disease-causing organisms biological control of postharvest diseases
continue to damage the fruit before and of fruits. The use of antagonistic
after harvest resulting in enormous microorganisms as an alternative to
losses. One of the most common fungicide under these conditions
pathogens attacking mango is Diplodia presents some unique opportunities to
natalensis Pole Evans, causing stem-end minimize or prevent pesticide residues
rot disease. This disease has been in fruits.
reported in Burma, Ceylon, Mauritius,
USA, India and the Philippines, with Research findings indicate that
infection of mango fruits from 2 to 6% Trichoderma spp. are of potential value for
(Halos and Divinagracia, 1970; PCARR, biocontrol of certain plant pathogens.
1975). Works dealing with biological control of
soil-borne plant pathogenic fungi often
Control of the stem-end rot disease report that Trichoderma spp. provide
of mango fruit involves both chemical significant reduction of diseases due to
and physical measures. The use of Sclerotium rolfsii and Rhizoctonia solani
benomyl (600-1000 ppm), dipping the (Chet, 1987; Maramara and Paningbatan,
fruits in 6% borax solution at 43C for 5 1993; Paningbatan, 1994). These
min and hot water treatment of fruit at organisms are known to act as
51-55oC for 10 min are commonly advised mycoparasites and produce diffusible
(PCARR, 1975). Hot water treatment, toxic substances and degrading enzymes
however, has some limitations. Beyond to other fungi in vitro (Lorito et al., 1993).
550C, the treated fruits get scalded or Although most biocontrol tests with
injured, while lowering the range from Trichoderma spp. were On pre-harvest
C- r~o/"" -1- _*_ _J"__*_ L _ 1 -f I

Philipp. Phytopathol. 1995, Vol. 31(2):103-116
post-harvest disease control. For
instance, Tronsmo and Dennis (1977)
found that the production of strawberry
fruits against storage pot (Botrytis cinerea
and Mucor mucedo) by spraying
aqueous suspension of conidia of
T. polysporum. In another study, Tronsmo
and Raa (1977) were also able to protect
apples against eyespot (B. cinerea) by
spraying conidia of T. pseudokoningii
after artificial inoculation of flowers With
the pathogen.

An isolate of D. natalensis was
collected from infected "Carabao" mango
fruit obtained from Cebu. Sections
of the infected fleshy portion of the fruit
were surface-sterilized by dipping in
0.5% sodium hypochlorite and water for

changes of sterile distilled water,
blotted dry in a sterile absorbent paper,
planted in petri dish containing
solidified potato dextrose agar (PDA)
and incubated in the laboratory.
Mycelial discs from the growth of the
fungus were cut and transferred
aseptically to PDA slants where they
were maintained.

Mass production of D. natalensis
was done on plated PDA. The medium
in each plate was seeded with an agar
block of the pathogen obtained from 2-
week old PDA cultures of D. natalensis.
Two-week old agar cultures of D.
natalensis served as the source of cordial
inoculum.

Stem-end rot development as affected
by inoculum levels of D. natalensis

Conidia from pycnidia crushed
aseptically in sterile water were used as
inoculum adjusted to four conidial
concentrations (5,000; 15,000; 30,000 and
50,000 conidia/ml). Ten microliters (ul)
of the different conidial concentrations of
D. natalensis were inoculated on the
exposed pedicel end of "Carabao"
mango fruits 3 days after harvest. The
inoculated fruits were then incubated in
previously disinfested 28 cm x 20 cm x 7
cm plastic boxes lined with sterile
moistened filter paper for a period of 8
days. Percentage rotting of the fruit
surface area and the volume of the fruit
infected with D. natalensis were visually
estimated. The concentration of conidia
which induced the most severe rotting
of the fruit and the methods of
inoculation and incubation were used in
thp sucrrelincr binasaav in vivo.

Different isolates of Trichoderma and 3 Antagonist and D. natalensis each
Gliocladium species used in the colonized approximately 1/2 of the
experiment were obtained from the medium surface (more than 1/3 and less
Department of Plant Protection, ViSCA, than 2/3);
Baybay, Leyte. These were: T. aureoviride
Rifai, T. viride Pers., T. harzianum Rifai, 4 D. natalensis colonized at least 2/
T. glaucum Abbot, T. pseudokoningii 3 of the medium surface and appeared
Oudem and other strains of T. glaucum to withstand encroachment by
f r o m antagonist; and
Dr. Delfin Lapis of the Department of
Plant Pathology, UP Los Bafos, College, 5 D. natalensis completely overgrew
Laguna. Additional fungal isolates were antagonist species and occupied the
isolated from the surface of newly entire medium surface.
harvested mango fruits taken from the
islands of Cebu and Levte. These isolates Effect of the most antagonistic funeral

controlling D. natalensis. An isolate of T. viride most
antagonistic to D. natalensis was tested
Agar discs were obtained from 2- to determine its effect on sporulation
week old plated cultures of the different capacity of the pathogen. Five-mm
isolates of fungal antagonists and D. diameter agar culture discs of both the
natalensis using a sterile 5-mm diameter antagonist and the pathogen were paired
cork borer. Agar discs of antagonist on plated PDA. After two weeks of
cultures were paired individually with incubation in the laboratory, the
that of D. natalensis on a previously sporulation capacity of D. natalensis
plated PDA. Paired agar discs were alone and D. natalensis + T. viride was
positioned 3-cm apart on plated PDA. examined using 1-cm diameter discs.
Five randomly sampled discs per
The treatments were replicated five treatment were each scraped gently to
times in a completely randomized collect pycnidia and these were crushed
design. Treatment means were compared in 1-ml sterile water in petri dishes to
using DMRT. After 5-14 days incubation, release the conidia. Mycelial fragments
the degree of antagonism against D. were separated by passing the
natalensis was measured using the rating suspension through 2 layers of nylon
scale developed by Bell et al. (1982): tulle. Conidial concentration was
determined using a haemacytometer
1 Antagonist completely overgrew and sporulation capacity was estimated
the pathozen and covered the entire based on a cm2 agar culture surface

Philipp. Phytopathol. 1995, Vol. 31(2):103-11

Bioassay of antagonism of bacterial
isolates against D. natalensis

Different isolates of bacteria used in
the experiment were isolated from the
surface of newly harvested mango fruits
collected from the provinces of Cebu,
Leyte and Rizal. These isolates were
bioassayed against D. natalensis in the
laboratory to determine the effective
isolate in controlling D. natalensis.

Conidia from pycnidia of D.
natalensis crushed aseptically in sterile
water were used as inoculum. One-half
ml of D. natalensis at 50,000 conidia/ml
of sterile water was placed into sterile
petri plates and then filled to one-third
with PDA culture medium.

One-day old slant cultures of the

cultures. One- to two-week old aga
cultures containing the fungal antagoniP
served as source of conidia fo
inoculation.

The different conidial concentration
of T. viride were determined using
haemacytometer. Two to three ml c
sterile water was poured on each slar
containing T. viride and the conidia wet
suspended in water using a Type 1670
mixer (Thermolyne corp., USA'
Conidial concentrations on the stoc
suspension were diluted to obtain th
desired concentrations: Inocula with th
different concentrations were placed i
flasks with one drop of Tween 80 adde
as surfactant per 20 ml.

Determination of the most effective
conidial concentration of T virid

scraped gently using a sterile wireloop. Fifty thousand conidia per ml of L
Five-mm diameter sterile filter paper natalensis found to be the most effective
discs were dipped in a cell suspension of conidial concentration of the pathoge:
each test bacterium adjusted uniformly was used in the study.
to 2 x 108 cells/ml and placed at the center
on a previously plated PDA with Three to five days after harvest c
D. natalensis. mango fruits, 10 ul of the aqueou
conidia were inoculated on the expose-
The treatments were replicated five pedicel end of the fruit immediately afte
times in a completely randomized spraying with different conidia
design. Treatment means were compared concentrations of T. viride. The treatment
using DMRT. The zone of inhibition were as follows:
against D. natalensis.was measured after To sterile water only
5 days of incubation period. T1 D. natalensis only
T2 104 conidia/ml of T. viride + E
Maintenance, mass production and natalensis
preparation of antagonist inoculum T, 10 conidia/ml of T. viride + E
natalensis
T. viride, the species found most T4 106 conidia/ml of T. viride + E
effective against D. natalensis in the natalensis
hiWccnx in 7,ifrn 1AC ,- cl ;n-Cn rw PA T 1 fl7 r.n;4;. /ml I-f T 7;r;,iA -L F

The treatments were replicated five
times and arranged in a completely
randomized design. The experiment was
done twice at ambient laboratory
conditions and incubated inside moist
plastic chambers as described earlier.
Proportions of infected surface area and
tissues affected in the mango fruit were
measured 8 days after inoculation.

Efficacy of T viride as affected by
inoculum levels of D. natalensis

Conidial concentration of T. viride,
108 conidia/ml, found to be effective in
controlling D. natalensis at 50,000

The treatments arranged in a
completely randomized design were
replicated five times. The set-up was
placed under laboratory condition.
Percentage of disease incidence on
mango fruit was determined 8 days after
inoculation.

Determination of the protective and
eradicative effect of T. viride on
D. natalensis infection

The most effective conidial
concentration, 108 conidia/ml, of T. viride
found in the previous test was used. The
source of conidia of D. natalensis was the
same as stated earlier.

Ten microliters of the aqueous

suspension of T. viride was applied each mango fruit one day after harvest in a
on ten injured sites of a partly ripened laboratory condition. The treatments
mango fruit. After the T. viride conidial were the following: mango fruit sprayed
suspension had dried, 10 ul from each of with T. viride conidia 5 days before
the different conidial concentrations of inoculation of D. natalensis; mango fruit
D. natalensis were inoculated on each sprayed with T. viride 3 days before
injured site. The treatments were as inoculation of D. natalensis; mango fruit
follows: sprayed with T. viride 3 days after
T, T. viride + 5,000 conidia/ml of inoculation of D. natalensis; mango fruit
D. natalensis sprayed with T. viride 5 days after D.
T2 T. viride + 10,000 conidia/ml of natalensis inoculation; and mango fruit
D. natalensis simultaneously inoculated with T. viride
T, T. viride + 15,000 conidia/ml of and D. natalensis.
D. natalensis
T4 T. viride + 20,000 conidia/ml of The treatments were replicated 5
D. natalensis times in a completely randomized design

Stem-end rot development as
affectedby inoculum levels of
D. natalensis

Disease incidence and severity of
stem-end rot in artificially inoculated
mango fruits were directly related to the
quantity of conidia of D. natalensis.
Inoculation of both 30,000 and 50,000
conidia/ml resulted in 100% incidence of
stem-end rot (Table 1). Although disease
incidence was highest in both
concentrations, inoculation of 50,000
conidia/ml caused consistently highest
fruit surface area and fruit volume rotten
at 85 and 97.7% respectively, which were
higher than the level of disease severity
due to inoculation with 30,000 conidia/
ml. Respectively, 77, 86 and 98% of

Table 1. Percent disease incidence and seven
8 days after inoculation of injured

Treatment Disease
(conidia/ml) Incidence

5,000 37 c

15,000 90 b

30,000 100 a

50,000 100 a

Control 0 d

CV (%) 3.95
r2 (%) 77

'Means with the same letter are not significantly difi

V .LI ILLU I If 11 An UI I L-1. I ILee CI I, .L -.I
surface area affected and fruit volume
rotten were explained by the variation in
the inoculum levels of D. iatalensis (Table
1). The diseased surface area of the
mango fruit was found to be less than
the estimated volume of fruit rotten upon
dissection. Therefore, colonization by D.
natalensis of the flesh was much faster
than of the fruit exocarp. Since it
consistently caused the highest level of
disease incidence and severity, the 50,000
conidia/inl was used in the succeeding
evaluation of biocontrol effectiveness.

Bioassay for antagonism of fungaland
bacterial isolates in vitro

Ten existing fungal isolates in the
Department of Plant Protection, ViSCA
and five new ones isolated from mango

ity due to infection by Diplodia natalensis
pedicel end of mango fruits'

Disease Severity
Surface Fruit Volume
Area Infected Rotten

30 c 38 d

47 c 62 c

57 b 78 b

85 a 97 a

0d 0e

17.12 12.42
86 98

rent at 5% level by DMRT

fruit surfaces were evaluated for
antagonism to D. natalcnsis using dual
culture method on plated PDA as
described by Bell et al. (1982). Twelve
bacterial isolates taken from newly
harvested mango fruits were also tested
for antibiotic effects on the stem-end rot
pathogen.

Among the 15 fungal isolates,
T. viridc consistently ranked as the most.
antagonistic to D. natalcnsis on two trials
(Table 2). The colony growth of

D. natalnisis on two dual cultures was
arrested and overgrown by T. viride.
Compared with the control, pycnidial
formation was inhibited when T. viride
grew over the pathogen. All the isolates
(Ml M5) collected from the surface of
the mango fruit generally exhibited least
antagonism to D. natalensis based on Bell
et al. (1982) rating scale. Earlier, T. viride
was found antagonistic to S. rolfsii
causing wilt in mungbean (Maramara
and Paningbatan, 1993) and R. solani and
P. debaryanlui causing damping-off and

d by the different fungal isolates against

Rating Mean
Trial 1 Trial 2

1.0 i 1.2 e 1.1
1.2 i 2.6 c 1.9

1.8 h 2.0 d 1.9

2.0 gh 2.6 c 2.3
2.4 fg 2.6 c 2.5
3.0 de 3.0 c 3.0
2.4 fg 3.8 b 3.1

2.8 ef 4.0 b 3.4
3.4 cd 4.0 b 3.7

Philipp. Phytopathol. 1995, Vol. 31(2):103-116

sheath rot diseases (Alcantara, 1987). The
observed antagonism of T. viride against
D. natalensis in the present study should
widen the spectrum of its biocontrol
applications. All the 12 bacterial isolates
taken from the surfaces of mango fruit
samples did not inhibit D. natalensis.

In addition to inhibition of
D. natalensis mycelial growth in dual
cultures, T. viride remarkably suppressed
the pathogen's sporulation capacity
(Table 3). In the absence of T. viride, D.
natalensis sporulated 32,500 conidia/cm2
of agar culture at 2 weeks of incubation
but this was reduced to 250 conidia/cm2
or reduction of 99%. Thus, T. viride was
used in the succeeding bioassays in vivo.

Bioassay In Vivo

Determination of the most effective
conidial concentration of T viride
against D. natalensis. Ten aqueous
conidia at five different concentrations
(104, 105, 106, 107, and 108 conidia/ml)

'Each value represents the mean of five
replications obtained from 1-cm diameter random
sample discs- means were compared using the
t-test.

were tested. Benomyl at 600 ppm was
included as fungicide check. The disease
parameters used in examining the
biocontrol efficacy were disease incidence
(Table 4) and disease severity in terms of
fruit surface area affected and fruit vol-
ume rotten (Table 5). Treatments of T.
viride at 105-108 conidia/ml significantly
reduced disease incidence compared to
the untreated control, thus, providing
levels of protection ranging from 33-80%
(Table 4). Application of T. viride at 10'
conidia/ml resulted in the lowest disease
incidence at 20% which was significantly
better than treatment with benomyl at
46.7%. Thus, the biocontrol efficacy of
80% obtained for T. viride at 108 conidia/
ml was better than the level of protec-
tion given by benomyl. In a related
study, however, T. viride at 109 conidia/
ml as seed coating suspension was found
to control wilt in mungbean better than
benomyl (Maramara and Paningbatan,
1993). Although the lower concentrations
can reduced incidence of stem-end rot in
this study, the aim in mango fruits is to
completely protect them from being in-
fected using the higher level of concen-
tration of T. viride. Fruits treated with T.
viride alone at 108 conidia/ml did not
show any observable lesion which indi-
cates that the biocontrol agent is harm-
less to the mango fruit.

The same pattern of levels of disease
severity was observed as in disease
incidence. Mango fruits treated with 105-
108 conidia/ml of T. viride significantly
reduced fruit surface area and volume
rotten compared to the control (Dn
alone), providing protection ranging
from 31.9 67.9% and 24.9 63.4%,
respectively (Table 5). Generally, a
significant decrease in the disease

Table 4. Percent disease incidence of stem-end rot due to inoculation with Diplod
niatalchsis as affected by prior treatments with different conidial concentration
of Trichoderia viride'

verity of fruit stem-end rot was
stainedd with the increasing
mcentration of T. viride against D.
italensis. This confirms the findings in
other pathogen-biocontrol agent
iteractions. McLaughlin and Wilson
992), for instance, showed that control
: post-harvest diseases of grape, peach
id apple is directly related to the spore
mcentration of the antagonist, D.
Italensis. However, the efficacy of T.
ride to protect the mango at 108 conidia/
.1 is comparable to the protection
trainedd by the mango fruit using
momyl at 600 ppm based on the two
disease severity parameters used.
application of T. viride at 104 conidia/ml
id not have significant control of rotting
S80 and 90% based on the fruit surface
tea and volume affected, respectively,
)mpared to the control.

Efficacy of T viride as affected by
different conidial concentrations of
D. natalensis Seven conidial concentra-
tions of D. natalensis (5,000; 10,000; 15,000;
20,000; 30,000; 40,000; 50,000 conidia/ml)
were used to examine the efficacy of T.
viride applied at 108 conidia/ml one hour
ahead of D. natalensis inoculation just
enough to air dry the applied aqueous
conidia of the biocontrol agent. Since
wounded exocarp can also serve as in-
fection court of D. natalensis (Halos &
Divinagracia, 1970), 10 injured sites/
mango fruit/replication was used.

The results indicate that biocontrol
efficacy of T. viride was inversely related
to higher inoculum levels of the pathogen
(Table 6). Complete protection was
attained when the pathogen inoculum
level was 15,000 conidia per ml or less.

:hoderma viride on disease incidence as
nt concentrations of Diplodia natalensis on

Relative
Tv + Dn Reduction
(%)

0 b 100
0 b 100
Ob 100
5b 95
5b 95
15 b 85
65 a 35
Ob

22.61
wound sites as affected by different conidial con-
ftpr arp nut -ihTificantlv different at 5% level by

When the D. natalensis inoculum
increased from 20,000 to 50,000 conidia/
ml, the level of biocontrol
correspondingly decreased. Within this
range the biocontrol efficacy declined
from 95 to 35% (Table 6). Indeed, it is
clear that when the disease pressure is
high the effectiveness of the biocontrol
agent to antagonize the pathogen can be
remarkably reduced. The results of the
study imply the need to determine the
natural levels of primary inoculum of
D. natalensis. If such levels will not go
beyond 150 conidia (10 ul of 15,000
conidia/ml) arriving as primary
inoculum in the infection courts (e.g.
stem-end, injured exocarp) then the use
of T. viride to fully protect mango fruits
looks very promising.

Protective and eradicative effects of
T viride on D. natalensis infection. In
order to optimize the application of T.

Table 7. Percent disease incidence and se
days after inoculation of Diplod,
application of Trichoderma viride1

Treatment Disea

Simultaneous Tv and Dn

Tv 5 days ahead of Dn

Tv 3 days ahead of Dn

Tv 5 days after Dn

Tv 3 days after Dn

CV (%)

'Inoculation of D. natalensis (Dn) was done on ex]
means with the same letter are not significantly d

pathogen, different periods of time ou
application of the biocontrol agen
relative to the inoculation time of D
natalensis were examined. T. viride at 10
conidia/ml applied before inoculation o:
D. natalensis had generally lower stem
end rot incidence and severity in term,
of fruit surface area infected than where
applied after (Table 7). This indicates tha
T. viride's action was more on protecting
mango fruits than on curing the disease
once it has already established
Histopathological studies have show
that D. natalensis establishes itself as earl3
as two days after inoculation (Halos anc
Divinagracia, 1970). Simultaneous
application of T. viride and D. natalensi:
consistently resulted in lowest disease
incidence (26.7%) and disease severity
(16.5%). The same pattern was observed
by Chalutz and Wilson (1990) in thei
work on D. hansenii against D. digitatun

verity based on fruit surface area infected :
a natalensis as affected by different times o

se Incidence Disease Severity

26.7 d 16.5 b

53.3 c 32.5 a

46.7 c 18.0 b

100.0 a 37.7 a

73.3 b 34.8 a

stem-end and T. viride (Tv) was spraye
% level by DMRT.

causing green moia on grapen'uit were
simultaneous and prior to inoculation
gave the better control of the disease.

Since T. viride's mode of action is
mainly protective in nature, its survival
after application needs to be investigated
thoroughly. This should enable us to
formulate measures that would enhance
the biocontrol activity by providing, for
example, the necessary food base along
with the applied inoculum.

LITERATURE CITED

ALCANTARA, T.P. 1987. Antagonistic
activities of Trichoderma species
against vegetable fungal pathogens
in vitro. BSA Thesis, University of the
Philippines at Los Bafios. 46 p.

Screenhouse test showed treatment of fluorescent Pseudomonas
during transplanting gave the lowest percent wilting of tomato plants
of 20 and 40%, with corresponding 77.3 and 57.4% control, for 294 x 108
and 251 x 106 cfu/ml, respectively. Lowest control was observed when
treatment was done 14 days after transplanting.

biocontrol agenf against tomato da
during transplanting.

INTRODUCTION

Tomato (Lycopersicon esculentum
lill.), a solanaceous vegetable plant, is
mne of the most profitable crops in the
'hilippines and is used in making
;ood source of vitamins A and C
/tm lr r '-nnr\

.. L t- vl/t-Ki .U L -1- -A -V ,
ing-off if applied in tomato plants

Tomato, like other fruit crops, is
subject to the attack of insects and
pathogens. Among the pathogens
nfecting tomato in the Philippines
and in other tomato growing
countries Rhizoctonia solani Kuhn the
:ausal organism of damping-off is
consideredd one of the most widespread

pathogens by introduced organisms has screenhouse.
been studied for over 65 years (Baker,
1987). In the early years, it has not been
considered economically feasible. MATERIALS AND METHODS
Currently, there had been a shift to the
opinion that biological control can have Laboratory Experiment
an important role in agriculture. This
renewed interest is in part a response to Preparation and maintenance o
public concern about hazards associated R. solaniand fluorescent Pseudomona,
with chemical pesticides. An isolate of R. solani was taken from th
culture collection of the Department c
Certain strains of fluorescent Plant Protection. The culture wa
Pseudomonas biologically control one or maintained by transferring bimonthly t,
more soil-borne pathogens when applied fresh potato dextrose agar slants an<
as seed-piece inoculants to agricultural incubated under ordinary laboratory.
crops (Schroth and Handcock, 1982). conditions.
These pseudomonads, referred to as
"plant growth-promoting bacteria A pure culture isolate of fiuorescen
(PGPB)" appear to displace or suppress Pseudomonas was maintained in nutrien
deleterious microorganisms or inhibit agar slants. Weekly transfer was don
pathogens that attack the roots. Several to maintain the viability of the isolates.
reports suggested that beneficial
Pseudomonas enhances plant growth and Determination of effective,
effect biological control by producing concentration of fluorescen

118

I A

ie Dacterium. ne plates were then each jar was seeded with agar block of
Lverted and incubated at 370C. The one-day old R. solani. The pathogen was
rowth of mycelia was measured after allowed to grow for two weeks prior to
1, 48 and 72 hr. Three replications were inoculation. This medium containing
lade for each treatment arranged in R. solani served as the source of sclerotial
)mpletely randomized design (CRD). bodies and mycelia for inoculation or soil
te plates were observed for mycelial infestation.
growth and those concentration that
lowed no growth were considered most Soil infestation with R. solani and
fective and used in the screenhouse planting of tomato seedlings. Two-week
:periment. old rice grain-rice hull pure culture of
R. solani were mixed with sterile rice
Determination of bacterial grains in the ratio of 1:3 (inoculum: rice
population. Serial dilution plating was hull) in a big container. One part of this
one to determine the number of mixture was then mixed thoroughly with
icterial population of the different nine parts soil. The mixture was then
lorescent Pseudomonas tubes with 25, transferred to pots. Then, ten tomato
i, 50 and 55 transmittance. The different seedlings were transplanted in the
altures were diluted from 10-1-10-1', after infested soil.
which one-ml sample from each tube was
aced into petri plates. Nutrient agar Treatment with fluorescent
as used as the medium for this test. Pseudomonas. The efficacy of fluorescent
after 48 hr of incubation, plates which Pseudomonas was tested using two
lowed 30-300 colonies were taken and bacterial concentrations namely: 25 and
e cfu per ml of the different dilution 50% transmittance. Except for To and T,,

Sterile water-treated tomato plants
grown in uninfested soil were provided
to detect natural infection of R. solani.
Each treatment was replicated five times
with ten plants per replicate and
arranged in CRD. Comparison of
treatment means were made according
to DMRT.

Data Gathered

Mycelial growth. This was
measured using a ruler by taking the
distance from the center of the petri
plates where the fungal inoculum was
placed up to the end of mycelial growth
of R. solani after 24, 48 and 72 hr.

Bacterial population. The
population of fluorescent Pseudomonas
was determined by getting the average
number of cfu per ml in each petri plate
after 48 hr of incubation.

Disease symptoms and percentage
wilting. Symptoms of the disease from
inoculation up to the first appearance of
infection was observed and described.
The number of plants wilted at five days
interval was recorded until the 25th day.
The percent wilting was computed based
on the formula: Percent wilting = No. of
Plants Wilted/Total No. of Test Plants x
100.

Results of the in vitro bioassay
showed that the different concentrations
of fluorescent Pseudomonas generally
suppressed R. solani. This is evidenced
by the complete inhibition of the mycelial
growth even after 72 hr particularly with
the bacterium at 294 x 108 cfu (Table 1).
The results showed that fluorescent
Pseudomonas had significant adverse
effect on R. solani. This finding was
consistent with the report of Kloepper
and Schroth (1981) that fluorescent
Pseudomonas exhibit antibiosis in vitro.
Moreover, Suslow and Schroth (1982) in
their study on sugarbeets concluded that
antibiotic production in vitro was
common to many rhizobacteria.

CV(%),)
Means of three replications; means in a column w
evel with DMRT

scalded appearance of circular to
irregular brownish lesions along the base
of the stem. The lesions expanded
upward from the point of inoculation
causingg necrosis along the whole stem.
Later, hyphal growth of the fungus
spread around the stem causing
extensive necrosis and consequently
withering.

Percent Wilting and Percent
Control. On the fifth day after
inoculation, tomato plants in the R. solani-
infested soil alone showed the highest
percent wilting, ranging from 28.0 and
30.0%. This was comparable to plants
'r -,f*Al TA7riTh flliln-crpnt PRaludmll ,n~ 14

1 II

mni as affected by fluorescent Pseudomnonas

Mycelial Growth-(mm)

24 hr 48 hr 72 hr

Oe Oe Oe

S10.67 d 12.33 cd 13.00 d

14.67 c 15.33 bc 16.67 bc

17.00 b 18.33 b 19.00 b

24.67 a 44.00 a 64.00 a

5.78 10.48 8.26
th similar letter are not significantly different at 5%

significantly different from those treated
with flourescent Pseudomonas 3 days after
transplanting. this trend continued until
the 15th day of observation. After this
time, no wilting was observed in the
plants treated with fluorescent
Pseudomonas at 294 x 10" cfu/ml and
251 x 10" cfu/ml.

Percentage of wilted tomato plants
varied when treated with flourescent
Pseudomonas at different times after
transplanting. The low value of 20 and
40% control was obtained with
inoculation during transplanting which
......... c1 \ -,, -.,- mrr; a ,4 Iuxr

122 r

flourescent Pseudoinonas at different periods of time relative to pathogen
inoculation'

Plants treated with flourescent
Pseudomonas during transplanting and at
3 days after transplanting provided
comparable level of protection which was
significantly better than those treated 7

(1986) mentioned that the rhizosphere or
rhizoplane must be occupied heavily by
the antagonist in order to achieve
protection for the disease to be controlled.
Therefore, pre-inoculation may be done
with the antagonist for it to grow and
fluorish prior to pathogen introduction.
In addition, biological control depends

of tomato seedlings treated with 294 x
eudomonas at different periods of time
ion.

124 Philipp. Phytopathol. 1995, Vol. 31(2):117-12

--R.. -+-R.e. fP P R... fP 3DAP
--

0

Philipp. Phytopathol. 1995, Vol. 31(2):117-126 125

upon the establishment and maintenance the suppression of native rhizosphere
of bacteria on planting material or in soil, microorganisms by plant growth
and a drop in viability may eliminate the promoting, fluorescent Pseudomonas.
possibility of biological control (Xu and According to Elad and Baker (1985),
Gross, 1986b). This may explain why fluorescent Pseudomonas can promote
lower percentage wilting was observed plant growth and disease suppressive-
in tomato plants treated at planting, ness through competition for carbon or
through production of hormones,
Between the plants treated with antibiotics and bacteriocins. Leong(1986)
fluorescent Pseudomonas at 294 x 10" cfu/ mentioned however, that compounds
ml and 251 x 10' cfu/ml, those treated other than antibiotics are involved in the
with the former exhibited lower percent biocontrol of pathogen by micro-
wilting than the latter. This is in organisms, of which the most widely
consonance with the finding of Dupler studied are the siderophores. Side-
and Baker (1984) which revealed that rophores are extracellular, low molecular
fluorescent Pseudomonas has the ability weight compound with a very high
to suppress root disease through affinity for ferric iron (Fravel, 1988).
colonizing it but has to be in great Siderophores produced by P. fluorescens
amount. Likewise, Mew and Rosales has the ability to sequester iron which
(1986) found that the presence of large provide a competitive advantage to
amount of Pseudoinonas spp. provides a microorganisms and play an active role
potential disease control of R. solani. of inhibition.
However, the population size of the
introduced bacteria may not be nearly as
important as the size of the population LITERATURE CITED
at the potential infection court. Generally
though, concentrations of introduced AVRDC. 1975. Asian Vegetable Research
bacteria at 10" per ml water when applied and Development Center, Tomato
to seeds or roots may affect other Report 51 p.
microorganisms in the rhizosphere which
BAKER, K.F. 1987. Evolving concepts of
often does not occur at lower dosages (104 biological control of plant
biological control of plant
per ml). pathogens. Ann. Rev. Phytopathol.
25: 65-85.
There are several reasons forwarded
for the control of Rhizoctonia by BECKER, J.O. and R.J. COOK. 1984.
fluorescent Pscudomonas. Howell and Pythiumi control by siderophore
Stepanovic (1979) have shown that the producing bacteria on roots of
production of antibiotic pyrrolnitrin was wheat. Phytopathology 74:806
the principal factor accounting to the (Abstr.).
control of Rhizoctonia in the rhizosphere DICKINSON, C.H. and J.A. LUCAS.
DICKINSON, C.H. and J.A. LUCAS.
of other seedlings. Kloepper and 1982. Plant Pathology and Plant
1982. Plant Pathology and Plant
Schroth (1978) also mentioned that Pathogens. (2nd ed.). 6: 72.
mutants of Pseudomonas have provided

ise of Acremonium wilt of guava
oduction system. Data suggested
!d during the second year and
! succeeding years of production
).288 per unit per year. The high
prompted farmers to discontinue
the 5th year of production.

wilt occurred in random foci and
regular manner along rows. This
* disease within the field was most
; operations.

d the destructive potential of
-ative production of guava in the

and defoliation of leaves and finally
death of the plant. Primary symptoms
include root rotting and brown to black
discoloration of the xylem tissues of the
roots and stems. A new species of
Acremonium tentatively identified as
Acremonium psidii was implicated to be
the causal agent of the disease. The
pathogen was shown to invade the

Lus oanos, louege, Laguna.

Keywords: Acremonium psidii, Acre
guava, Psidium guajava

AB

The temporal and spatial in
was documented in a commercial
that the disease normally occ,
progressed very rapidly during
with an estimated infection rate
destructive potential of the disei
the production of guava as early

Spatial spread of Acremonii
the disease apparently spread ir
indicated that the rapid spread of
likely mediated by frequent prui

The study had demonstr
Acremonium wilt to curtail the
Philippines.

INTRODUCTION

Acremonium wilt of guava (Psidium
guajava) was first reported in the
Philippines in 1984 by Quimio et al.
(1984). The disease was regarded as very
destructive wilt disease characterized by
wilting of leaves and young shoots in
certain branches followed by yellowing

In the later part of 1980's, several
hectares in Southern Tagalog Region
were planted to guava from Thailand
(locally known as Guapple) to meet the
demand in Metro Manila. However,
most farmers abandoned .their guava
farms after 3 to 4 years of production
primarily due to Acremonium wilt.

This study was conducted to
document the epidemic development of
the disease under local guava production
systems in order to provide insight for
logical and efficient management of the
disease.

MATERIALS AND METHODS

The Experimental Site

A one-hectare guava farm in
Barangay Siranglupa, Calamba, Laguna
was used in the experiment, The area
previously planted to sugarcane was
deep plowed (subsoiling) and harrowed
twice during the dry season. At the onset
of wet season (May) two-month old
seedlings of guava (cv Guapple) were
planted at a distance of 3 x 1.5 m. Weed
management was done primarily by the
combinations of cultivation (ring
weeding), use of power grass cutter and
chemical spray with glyphosate (Round-
up). Complete fertilizer (14-14-14) was
applied around the plants at the rate of
60 kg NPK/ha during the first year and
was increased to 120 kg NPK/ha during
the succeeding years. The fertilizer was
usually applied in three splits first,

Philipp. Phytopathol. 1995, Vol. 31(2):127-131
during the onset of rainy season (May),
second, during fruit development
(August), and lastly, at harvest time
(November). Plants were frequently
pruned to come up with desired canopy
crown and to induce flower bud
formation. Fruits were thinned out to
maintain a uniform fruit size. Insect pest
damages were minimized using frequent
insecticide sprays and bagging of fruits.

Monitoring of Acremonium Wilt

The development of Acremonium
wilt was monitored in time and space.
This was done by preparing a planting
plan of the whole area to indicate the time
and exact locations of infected guava
plants. Wilted plants were immediately
removed and vacant hills were replanted
with papaya (cv Solo). The spread of the
disease in time and space was
summarized at the end of each growing
season. The disease epidemics was
analyzed using logistic model (van der
Plank, 1963).

RESULTS AND DISCUSSION

The temporal and spatial increase of
Acremonium wilt of guava in a local
production is presented in Table 1,
Figures 1 and 2. The disease appeared
during the second year and progressed
very rapidly in the succeeding years of
production. At the end of 5th year, the
disease incidence reached 38.6%. Using
a logistic model, the rate of infection was
estimated at 0.288 per unit per year. The
production of guava was discontinued
in the 6th year due to the high expected
incidence of the disease and the
uncertainty of economic production.

Philipp. Phytopathol. 1995, Vol. 31(2):127-131 12I

Table 1. Incidence of Acremonium wilt of guava in a commercial production system

Cumulative No. Cumulative
Year of Infected Plants Disease Proportion

1989 0 0
1990 176 0.163
1991 223 0.206
1992 278 0.257
1993 417 0.386

Total No. of Plants 1081

0.3

0.2

YEAR

Figure 1. Disease progress curve of Acremonium wilt of guava in commercial
production system

Banana meriplants produced
throughh tissue culture were tested for
suitability as material for screening
Fusarium Wilt resistance. The reaction
Af the meriplants after inoculation with
knownn isolates of Fiisariumi oxysporuin f.
sp. cubense was first compared with Musa
ialbisiana seedlings. The disease reaction
Af the test meriplants was evaluated after
50 days by scoring the ab6ve ground
symptoms using a rhizome discoloration
ndex. The number of discolored
-hizomes from which F. oxysporuin f. sp.
'ube'nsc could be re-isolated was used as
;he basis for percentage infection.

The results of the inoculation
experiments established the standards
or evaluating the resistance of candidate
clones when inoculated with known
solates of F. oxysporuin f. sp. cubense. The
suitability of banana meriplants for
screening of Panama Disease resistance
ias been verified with all the inherent
advantages of handling smaller plants.
The standard procedure used-meriplants
it the 6- to 8- leaf stage which were
transplantedd into highly infested soil in
Lfirvi Cr kfr l T-I^- t- pir-*- ow ln nho / i pl hnr r riC\ fr el7

The clones showing no rhizome
discoloration were advanced to the
secondary test in cement beds infested
separately with'identified virulent
isolates. The plants were evaluated after
another 60 days. Plants that were not
infested in the secondary test were
multiplied and tested simultaneously in
the concrete culverts with sealed bottom
and under field conditions. The plants
were subjected to a broad collection of
isolates in the culverts while their
agronomic and commercial potential
were being observed in the field. A 4-
fold increase (35%) in the number of
uninfected plants were obtained from the
populations that were advanced to the
secondary screening. Four clones were
identified as highly resistant in the
secondary screening which were
advanced to field testing.

A collection of banana varieties was
established adjacent to two commercial
banana plantation in Davao del Norte
and Bago-Oshiro, Bureau of Plant
Industry Experiment Station, Mintal,
Davao City. These collections were tested
for Panama disease resistance. Priority
was given to varieties with commercial
npotentials.

rnlipp. rnytopathol. 1995, Vol. 3](2):132-144

Preliminary observations of the
varietal reaction to virulent isolates of
Fusarium oxi/sporumi f. sp. ciiubcs were
conducted using meriplants planted in
12-inch clay pots in the greenhouse.
Evaluation were based on the disease
severity index and the rhizome
discoloration index taken 60 days after
inoculation. Varieties exhibiting
resistance were planted around newly
eradicated infected mats in the field. The
varieties were considered susceptible
when they become infected. Those that
remained uninfected were multiplied
and planted in additional sites.

The experiment identified 41 highly
susceptible varieties. Among the
AAAs, Ipod'and a Roy Kerr accession
have shown resistance to Panama
disease. The ABBs were observed
resistant under field conditions. Of the
ABBs, only Latundan Tsina had shown
resistance in the greenhouse tests. Of the
AAs, Seflorita, Inarnibal, Pisang Lilin and
Pisang Kelat have shown moderate
resistance to immune reaction when
suckers were inoc.,l.:1ed in the
greenhouse.

The Occurrence, Morphology and
Pathogenicity of Fusarium o,ysporum
f. sp. vasinfectum in Cotton. Lydia C.
Cano and Aida D. Solsoloy, Cotton
Research and Development Institute
(CRDI), Polomolok, South Cotabato.

The occurrence, inorphologyv and
pathogenicity of fusa7riiuml ox/!sjiorum f.
sp. vasinif'ctuml was studied from August
1989 to February 1991, in the CRDI
Mindanio Area" Research Center;
Polomolok, South Cotabato.

I ne disease occurred in Klinan "> and
Conel, General Santos City, and in Klinan
6 and Silwav 7, Polomolok, South
Cotabato. Highest infection was noted
in Conel, General Santos City, and least
in Silway 7, Polomolok, South Cotabato.

Morphologically, the pathogen has
white and septated mycelia, produced
macro- and micro-conidia, sclerotia and
intercalary and terminal chlamydo-
spores. It produced violet pigmentation
in PDA and PSA media.

Pathogenicity test showed higher
infection at 25,000 spores/ml by stem
inoculation. The characteristic symptoms
and identity of the pathogen were the
same for the naturally infected and
inoculated plants.

Farmers' rice seeds were collected
from dry season (56 seedlots) and wet
season crops (64 seedlots) in 1993 from
Cavite, Laguna and Quezon provinces.
Seed health evaluation was done using
the between paper method followed by
blotter incubation of abnormal seedlings
and dead seeds to elucidate infection
causing germination failure on dead
seeds and seedling decay on abnormal
seedlings. Seven ranges of seed infection
were tentatively established from 0 to
above 40%. Majority of the seedlots had

134
both seasons. Dry season grown seeds
had a maximum of 23% seed infection
while wet season seedlots had infection
up to 49%.

The prevalent fungi was Fusariuin
moniliforme with maximum infection of
17, 49 and 32'% for Cavite, Laguna and
Quezon areas, respectively. Lower
infection of F. moniliforme were observed
in the dry season with 17, 5 and 23% for
Cavite, Laguna and Quezon,
respectively. Other Fusariuin species
were: F. solani, F. equiseti, F. semitectum, F.
avenaccum from Lucban, Quezon (dry
seasons), and F. larvarum from Cavite
area (both seasons). Trichoconis padwickii
(stackburn) and Curvularia spp. (black
kernel) had higher infection in the dry
season while Dreschlera oryzac (brown
spot) is relatively higher in the wet
season. Other fungi with lower
incidence causing death of seeds or
seedlings were Penicillium sp., Aspergillus
sp., Rhizopius sp. (generally higher in the
dry season), Gerlachia oryzae, Phoma sp.,
Penicillifer pulcher, Sarocladimn oryzac,
Verticilliumi cinnabarinumti and Tilletia
barclayana.

Survey, Isolation and Characterization
of Agrabacterium spp. in the
Philippines. Nenita L. Opina, J.E. Villa
and.C.M. Protacio, Institute of Plant
Breeding and Department of
Horticulture, University of the
Philippines Los Baios, College, Laguna.

A disease showing tumor growth
symptom on the stem and crown
portions of roses (Rosa sp.) was observed
in four provinces of Luzon, namely,
Benguet, Camarines Sur, Cavite and
l.nauna. The causal organism was

Philipp. Phytopathol. 1995, Vol. 31(2):132-144
isolated from the infected tissues of the
plant and was identified as Agrobacteriu m
tumefaciens. Pathogenicity tests using
tomato seedlings showed that eight
isolates caused gall symptoms.
Biochemical tests reveal that the isolates
belong to either biovar 1 or biovar 2.

Seeds from papaya fruits (cv Solo,
Cavite Special and Sinta) showing typical
concentric ringspot symptoms on their
peel were assayed for the presence of
papaya ringspot virus (PRSV) using a
modified enzyme-linked immuno-
sorbent assay (ELISA).

Results of assaying intact individual
seeds revealed 11-14% seeds tested
positive against the specific PRSV
antiserum. Likewise, different seed
parts, namely, sarcotesta, liquid, seed
coat, endosperm and embryo, and
coleoptyles from growing-on-tests were
also assayed. Data from both tests
suggest that the PRSV isolate observed
in the Philippines attest to the seed-borne
but not seed-transmitted nature of the
virus.

One hundred twenty-three (123)
Xanthotnonas cainpeistris pv. di'ffi'nbachiae
(Xcd) isolates were collected around.Los
Bafios, Laguna and tested in vitro for
comparative reactions and charac-
terization. The heterogenous nature of
the bacterium was determined by
studying the capacity to hydrolyze
starch, streptomycin resistance and
serological reaction to a monoclonal
antibody.

Among these isolates, 33 were
resistant to 50 ppm streptomycin. From
these 33 isolates, 29 were resistant to 200
ppm streptomycin. Further tests showed
that 17, 16 and 12 isolates were also
resistant to 500, 1000 and 2000 ppm
streptomycin, respectively. Fifty-eight
(58) isolates were able to hydrolyze starch
while 65 isolates were unable to
hydrolyze starch. The starch hydrolyzer
isolates grew rapidly on starch agar
medium compared to non-starch
hydrolyzer isolates that grew slowly in
the medium.

Mab Xcd 108 had reacted with 62 out
of 123 Xcd isolates based on indirect
ELISA at 410 nm. It was evident that
differences in bacterial populations were
associated with particular cultivar and
locality or farms.

In 1993, White Root Rot (WRR)
disease of rubber was monitored in four
regions comprising five provinces of
Mindanao. Of approximately 72,000 ha
of rubber plantations surveyed about
85% exhibited symptoms typical of white
root rot infection. The extent of infection
ranged from 15.31 to 46.82%.

The presence of the disease carr be
seen through the characteristic yellowing
of the leaves, die back of twigs and bran-
ches, rotted roots, fungal fructification at
the collar of the tree and dead trees.

Highest percentage infection
(46.82'%,) was noted in Bukidnon prov-
ince, particularly in the municipalities of
Damulog, Dangacagan, Don Carlos,
Musuan and Kibawe while moderate in
the provinces of Agusan del Sur, Davao
del Norte, and Zamboanga del Sur, with
an average infection of 25.33, 21.41 and
20.10%, respectively. The lowest disease
infection (15.31%) was observed in the
province of North Cotabato:

Surveys were conducted in local
sweet potato fields to assess the incidence
and distribution of sweet potato
feathery mottle disease. Transmission
experiments were done in the
screenhouse to determine the mode of
transmission of the virus.

136
Virus disease incidence in ViSCA
ranged from 2.9-13.8%. Severity of the
disease varied between accessions and
seasons. An increase in disease incidence
was observed during the wet season. In
the PRCRTC sweet potato germplasm,
out of 236 accessions, 21% were infected
with sweet potato feathery mottle.

In the screenhouse, the virus is best
transmitted by grafting and by aphids,
with transmissibility ranging from 60-
80% and 50-80%, respectively.

Evaluation of Different Artificial Media
for the Growth and Reproduction of
Phytophthora colocasia. M.K. Palomar,
V.C. Mangaoang and V.G. Palermo,
Department of Plant Protection, Visayas
State College of Agriculture, Baybay,
Leyte.

Three artificial media, namely, onion
agar, V8 juice agar and potato dextrose
agar were used to determine where
Phytophthora colocasia would best grow
and reproduce. An average of 85.8, 75.2
and 60.7 mm in mycelial growth
diameter of P. colocasia were obtained in
V8 juice agar, onion agar and PDA,
respectively. P. colocasia grown for 4-7
days in onion-agar gave abundant
sporangia after an agar disc of mycelia
was transferred to replacement liquids
(sterilized tap water and distilled water)
for 2-3 days under laboratory condition.

Philipp. Phytopathol. 199.. Vol. 31(2):132-144
The effects of different types of
medium, pH, exposure to light and
temperature on conidial production of
Cercospora kikuchii were studied.
Abundant in vitro sporulation of the
fungus was recorded in mungbean seed
decoction agar followed by soybean leaf
decoction agar and V-8 juice agar after
14 days of incubation.

Highest sporulation was noted at
pH 6.0-6.6, at 25"C, exposure to 12-hr
light and 12-hr darkness daily and lowest
sporulation in soybean seed decoction
agar, pH 7.5 at 250 C and at 350C at pH
6.5 under continuous light.

The effects of soil solarization,
planting method and varietal resistance
on tomato bacterial wilt incidence were
determined in an area with very high
incidence (60-100%) of bacterial wilt.
Fifty'percent of the plots were covered
with plastic sheets. Uncovered plots
served as control. The two planting
methods were direct seeding and
transplanting. Test plants were Yellow
Plum, a highly susceptible variety, and
VC-11-1, a moderately resistant variety.

From May 2 to June 16, 1994, soil
temperature in plastic-covered plots
ranged from 43-62"C with a mean of 50C
while the temperature in uncovered plots
ranged from 37-48"C with a mean of
40.3'C. In general, lower incidence of

Field trials were conducted during
Le dry and wet seasons of 1994 at the
RRI experiment station to determine the
fect of two potential biological control
;ents (BCA) on the focal expansion of
ce sheath blight. Each of the two
olates, In-b-6854 (Pseudoimonas cepacia)
id In-b-1821 (Pseudomnonas putida) and
mixture of both were applied to seeds
id were sprayed on transplanted IR 72
[ants. Sterile water and fungicide
inggamycin) were used as control.
elected spots of a plot were inoculated

137

tillering stage. Sheath blight severity and
spread of the disease were assessed at 0,
7, 28 and 42 days after inoculation.
During the dry season, suppression of
focal expansion in BCA-treated plots
ranged from 11.36 to 46.15% at panicle
initiation, 0.19 to 43.22% at flowering and
0.30 to 17.29% at harvest. Disease
severity was lowest in the plot treated
with the BCA mixture and yield was also
significantly higher in treated plots as
compared to the control. In the wet
season, variation in focal expansion and
yield was influenced by the type and
timing of BCA application. In general,
disease spread was lower in all plots
treated with single isolates or mixtures
of the BCA as compared with those
which were untreated.

Incidence of high infection of
Aspe'rgillus spp. and Penicillium spp. was
observed on seeds of ampalaya
(Momordica charantia L.). These
pathogens caused low germinability of
the seed rendering it unsuitable for
planting.

138 P
Under in vivo trial, pure extract and
75% concentrations from four test plants
were inhibitory to the growth of A.flavus
in an artificial medium. Panibat leaf at
25% concentration manifested antifungal
property against A. flavus. 'Bauang-
bauangan at 50, 75 and 100%
concentrations outperformed Captan
(positive check) in inhibiting mycelial
growth of A. niger in an agar medium.
The effectiveness of Captan in
suppressing fungal growth was
comparable only at 25% concentration of
Bauang-bauangan and 100%
concentration of Panibat. A. niger
mycelial growth was moderately
inhibited by Kalachuchi leaf extract at 100
and 75% concentrations and Kamantigue
leaf extract at 100, 75 and 50%
concentrations.

The in vivo experiment revealed
that Bauang-bauangan leaf extracts at
100, 75 and 50% concentrations were
effective in controlling A. flavus and A.
niger infection on seeds during a one
month storage time. Bauang-bauangan
leaf extract at 100, 75 and 50%
concentrations also minimized the
rapid loss of germinability of seeds
during one month storage. Only
Bauang-bauangan at 100, 75 and 50%>
were comparable with that of the positive
check, Captan.

Erwinia sp., isolated from club roots
of cabbage was identified based on mor-
phological and biochemical characteris-
tics. It has biological control potential

against Plasmodiophora brassicae, the
causal agent of clubroot on the brassicas.

Drenching Erwiiiia sp. suspension to
pechay plants six days after transplant-
ing markedly reduced clubroot severity.
However, drenching of said suspension
does not differ significantly with drench-
ing immediately after planting, dipping
of seedlings in Erwinia sp. suspension for
30-45 min, and using perlka lime at the
recommended rate.

The biological control activity of
three isolates of Trichoderma harzianum
from the Philippines against Sclerotiun
rolfsii, the causal agent of seedling blight
of rice, was investigated under field con-
ditions.

Inoculum of S. rolfsiiwas prepared
on sorghum seeds enriched with molas-
ses and complete fertilizer. Trichoderma
inoculum was prepared on rice hull and
bran mixture enriched with molasses and
complete fertilizer. The Trichoderma in-
oculum was applied at sowing time
along the furrows of the soil artificially
infested with S. rolfsii.

Treatments with any of the three Tri-
choderma isolates showed a decrease in
seedling blight incidence and severity; an
increase in plant-height 30 days after
sowing and at harvest with reduced
number of unproductive tillers; an in-
crease in number of filled grains; and

hilipp. Phytopathol. 1995, Vol. 31(2):132-144

)00 grain weight and total yield. Yield
crease was similar to that of the
enomyl-treated control plot.

fficacy of Trichoderma sp. as
biological Control Against Fusarium
xysporum f. sp. vasinfectum. Lydia
. Cano and I.G. Catedral. Cotton
research and Development Institute,
2RDI), Polomolok, South Cotabato.

The study was conducted at the
RDI Center Experiment Station,
olomolok, South Cotabato from
september 1993 to April 1994 to evaluate
ie efficacy Trichodenna as a biological
)ntrol against Fusariun oxysporun f. sp.
isinfectuin.

The efficacy of Trichoderma sp. was
evaluated through bioassay and in situ
sing the following concentrations
),000; 100,000; 200,000; 400,000; 800,000;
,600,000 and 3,200,000 spores/ml. The
treated check was sterile distilled
'ater. The effective concentration
.00,000 spores / ml) was further verified
i the field.

Results showed that Trichodermna
issolved all the mycelia of Fusariumi at
5 days after isolation.

139

Trichoderma and drenched four times at
10, 20, 30 and 40 days after planting,
which was comparable to the disease
control provided by the chemical
fungicides.

Effect of the Rate of Carbon, Nitrogen
and pH of the Substrate on Fruiting of
Oyster (Pleurotus flo'ida) Mushroom.
B.S. Saya-An and B.S. Tad-Awan,
Benguet State University, La Trinidad,
Benguet.

This study was conducted to find
out the levels of carbon, nitrogen and pH
of the substrates currently being used for
oyster mushroom, indigenous farm
wastes and to assess the return on
investment.

Result showed that substrates
currently used had 8.12 to 25.5% C; 0.805
to 2.205% N and pH 5.2 to 7.3.

Carbon level at 17.86, nitrogen
level at 1.54% and pH 5.6 enhanced
the highest yield with the use of rice
straw as the main substrate and 15%
rice bran and sugar as additive.
Conversely, carbon and nitrogen at
undetermined level and pH 6.4 effected
the lowest yield.

T_4r 4..4- C 1 3

IlUClIIlUjIU l l UUill UII U IJlV IIV- V LILI l CLi'Il LiUA . Y xuU, -
a Production of the Rice Sheath Blight. Laguna.
A. Elazegui, N.P. Castilla and S.
vary, International Rice Research In- Isolates of Rhizoctonia solan
itute (IRRI), Los Bafios, Laguna. collected from different areas of
and Mindanao. Hyphal fusion b(
Sclerotia of Rhizoctonia solani were testers and isolates of R. solani frn
illected from infected plants in lowland, ferent crops were detected to det
rigated ricefields of Batangas, Bulacan, their anastomosis group type.
iguna, Nueva Ecija, Quezon, and Rizal
October 1994. Sclerotia collected from Results showed that all isolat(
ricefield in each province was consid- corn, sorghum, wheat, mun
ed as an isolate. Sclerotia production aguingay and sugarcane belong ti
r the different isolates as affected by IA and they cross infect with co
oculum amounts and leaf wetness du- sorghum. Isolates from potato be
tion was then studied in screenhouse. AG-3 while isolates from ca
*lerotia from an isolate collected in IRRI, matched with AG 2-1. The isol
which has been used as inoculum for AG 2-1 and AG-3 were mainly cc
,periments, was included, from the cool areas of Baguio anc
cinity and pathogenic only to poti
Bulacan isolate significantly pro- crucifers, respectively.
aced the most number of sclerotia fol-
wed by Batangas and Nueva Ecija iso- Symptomatology of Mulberry R1
tes, with average counts of 52, 27 and the Morphology of its Patl
;, respectively. The IRRI isolate pro- Aecidium mori Barclay. Teod
uced the lowest number of sclerotia, Dizon and M.M. Kakishima, Ir
ith an average count of 3. Sclerotia pro- of Plant Breeding, University
auction was not affected by inoculum Philippines Los Baiios, College, 1
nount (5 vs. 15 g) nor by leaf wetness and University of Tsukuba, Tsi
duration (wet every night, wet every Ibaraki, Japan.
:her night or completely dry). For all
olates, the number of sclerolia formed The occurrence of mulberry r
creased over time. Information on this reported to occur for the first I
hase of the sheath blight cvle will be Zamboanga City. Symptoms
rnthesized with data on other phases disease consist of formation of <
id with other epidemiological data in a bright yellowish orange aecia ak
mulation model, thereby providing veins and lamina of the affected 1I
leans to help delineate management portion of the stem, petiole, pedun
rategies for sheath blight, flower. On stem, affected portion I
hypertrophied. Aecia hypophyl
nastomosis Group Type of Rhizocto- epiphyllous, erumpent, sub-epi
ia solanifrom Different Host Species. scattered or aggregate; peridi&

Philipp. Phytopathol. 1995, Vol. 31(2):132-144 141
developed; aeciospores globose to College, Laguna, International Rice
ellipsoid, verrucose, catenulate. Research Institute, Los Bafios, Laguna
and Philippine Rice Research Institute,
This study is the first attempt to Mufioz, Nueva Ecja.
describe morphologically the causal
pathogen of mulberry rust in the A 3-season crop sequence study,
Philippines. involving onion, garlic, mungbean, and
sorghum, with the objective of finding
Effects of Flooding Time, Water out whether sheath blight is suppressed
Regimes, and Soil Types on Yield of after an initial rice cropping inoculated
Different Rice Cultivars Infected with with Rhizoctonia solani, was undertaken
Rice Root-knot Nematode, at the PhilRice experiment station in
Meloidogyne graminicola. Imelda R. Mufioz, Nueva Ecija. Based on sheath
Soriano, D.M. Matias, and J.C. Prot, blight incidence on rice cultivar IR72
International Rice Research Institute, during the third season, significant
Los Bafios, Laguna. differences were observed among
treatments with onions showing a
In the first experiment, UPLRi5 particularly suppressive effect.
grown on sandy and clay loam soil
inoculated with Meloidogyne graminicola In another study covering three
(Mg) was flooded at different growth seasons involving cultural practices
stages. In another experiment, IR20, including sanitation and fallowing, no
IR24, IR72, and IR74 were grown in two significant differences were observed
soil types, clay and sandy loam, under among treatments in terms of sheath
flooded and rainfed conditions to study blight incidence. It indicates that a longer
the effects of Mg on yield. period of time is needed for cultural
practices such as those aforementioned
Yield of UPLRi5 grown in sandy to show significant effects.
loam soil was significantly reduced (P<
0.05) in all period of flooding. In clay A study on the effect of soil types,
soil, yield of UPLRi5 was not affected collected from different regions in the
under continuous flooding but was Philippines, on R. solani showed
significantly reduced under rainfed significant differences in sclerotial
condition. However, in clay soil, only the viability. Chemical analysis revealed that
yield of IR24 was significantly reduced loam soil types decreased survival while
upder rainfed condition. The yield of clay and clay loam types exerted
IR72 and IR74 were not affected by Mg favorable effects. High Zn content was
infection in all water regimes and soil found associated with decreased survival
types. while high P and high Mg were related
to increased survival.
Cultural Practices and Epidemics of
Sheath Blight of Rice. A.D. Raymundo, Quantifying the Effect of Soil
M.R. Pantua, A. Baria, and P.S. Teng, Amendments on the Development of
University of the Philippines Los Bafio4 Sheath Blight of Rice. M.R. Pantua, A.

142 Philipp. Phytopathol. 1995, Vol. 31(2):132-144
D. Raymundo and P.S. Teng, down the stalk. At the early stage of
International Rice Research Institute, wilting, the roots of infected plants
Los Bafios, Laguna. appeared normal except for few
secondary roots with black tips. Most of
The effects of animal manure, the diseased plants displayed disking of
organic fertilizer, and Sesbania rostrata as pith.
soil amendments on the development of
sheath blight on rice cultivar IR72 and The fungus isolated from infected
on mungbean were quantified. In a trial plants had morphological characteristics
using IR72, animal manure significantly similar to Phytophthora spp. The
suppressed sheath blight development pathogen induced black stem rot within
based on relative lesion length. Lower 3-4 days using stem inoculation method
infection rates were also obtained in the on 70-day old tobacco seedlings.
animal manure treatment when
compared to the organic fertilizer and S. An isolate of the fungus taken from
rostrata treatments. The rates of soil samples collected in Misamis Oriental
amendment showed significant was also pathogenic on tobacco.
differences while the level of inoculum
applied exerted no discernible effect. Factors Influencing Postharvest Disease
With mungbean, the soil amendments Resistance in Taro. C.S. Del Rosario and
significantly reduced disease severity as A.P. Molato, Philippine Root Crop
compared to the no-amendment Research and Training Center, Visayas
treatment. Soil amendments, likewise, State College of Agriculture, Baybay,
caused significant reduction in the Leyte.
viability of sclerotial bodies recovered
from the soil. Two varieties, VG-1 Kalpao and VG-
2 (Iniito), which have consistent resistant
Tobacco Blank Shank Incidence in and susceptible reactions to three major
Ilocos Norte: Luzviminda R. Truong postharvest diseases, Botryodiplodia
and Menisa M. Agpoon, National theobromac, Sclerotium rolfsii and Fusarium
Tobacco Administration, Batac, Ilocos soloni, respectively, were used in the
Norte. study.

In crop year 1993-94, reports of Results on the effect of root extract
serious tobacco wilt problems in Ilocos from resistant and susceptible taro
Norte were investigated. Total crop varieties showed greater growth
failure resulting from death of plants inhibition of the three pathogens in
prior to harvest and very poor transplant extracts from infected resistant tubers.
recovery were noted in some farms in the Likewise, different types of injury
municipalities of Dingras and Badoc. significantly affected the shelf-life of both
resistant and susceptible varieties.
The diseased plants had dark brown Tubers with the surface rubbed with
to black necrotic stem. Necrosis started coarse sand paper were the first to show
from the soil line and extended up and outward infection followed by tubers cut

with surgical knife at the root-end Thirty RAPD primers were used to
portion. In addition, the shelf-life of taro amplify the DNA of different isolates of
can be prolonged by refrigeration. Thus, P. solhnracearmin. The apparently unique
the resistance of postharvest diseases in DNA fragments from each strain were
taro is greatly affected by temperature isolated, labelled with "P and used as

. tubers.

rence of Peanut Stripe and
es in Peanut in Marin
Ila M. Barcial, N.B. Bajet a
ral, Department of
logy, University of the Phil
aiios, College, Laguna.

he peanut growing ai

cted genomic DNA
cearunM Probes showing
lization were directly do
k cloning site of PCR 11 T?
ed clones were sequence
ird and reverse prime
hoped. The developed prirr
to amplify genomic DP
icearuin and other Pseu
es to test their specificil

detected using indirect ELISA and applied to P. solanacearuin UNA. Ut the
membrane immunobinding assay. Most 7 probes tested, OPD-2 probe obtained
of the leaf extracts from all growing areas from biovar 4 was selected because it
positively reacted to the PStV and PMV hybridized to an apparently identical
antisera indicating the occurrence of both bands in ECO R1 digested DNA of P.
viruses in several peanut growing areas solanaccarinm. The forward and reverse
of Marinduque. primers were derived when the insert of
the 092-2B clone was sequenced. The
Development of Probes and Primers for sequence of the forward primer 759 was
the Detection of P solanacearum. 5'GTC GCC GTC AAC TCA CTTTCC 3'
Nenita L. Opina, J.N. Timmis, M. Fegan while the reverse primer 760 was 5' GTC
and A.C. Hayward, Institute of Plant GCC GTC AGC AAT GCG GAATCG 3 .
Breeding, University of the Philippines The developed primers produced a
Los Bafios, College, Laguna; single invariable 281 bp PCR product in
Department of Genetics, University of all the biovars of P. solanacearumn
Adelaide, South Australia and including Moko and Bugtok of banana
Department of Microbiology, but not P. pikettii and P. andropogonis.
University of Queensland, Brisbane, Sensitivity test showed that these
Australia. primers can detect approximately 1 cell

144 rninpp. rnytoparnot. Ia, vol. aiJ -i:J.-J
of P. solanrccarmi in a PCR reaction. in experimental farms anid greenhouse
of Benguet State University during t
Performance of silkworm (Bombyx rainy season (June to September) of 19
mori) Fed with Mulberry Leaves and1994. Diseasedplantswerecollecte
Sprayed with Various Fungicides. Luisa their symptoms described and examine.
B. Bugnay, A.M. Inumpa and B.S. Tad- microscopically for associated path
Awan, Benguet State University, La gens. Pathogens were isolated using a
Trinidad, Benguet. propriate media and isolation tec
niques. Pathogenicity of isolates w
Mulberry leaves sprayed separately tested on healthy greenhouse-grov
with four fungicides (Bayleton, Rover, chrysanthemum m using appropriate i
Vondozeb and Saprol) were fed to silk- oculation techniques.
worms and were assessed for their toxic
effect on the silkworms. Leaf spots caused by Altcrnaria sI
crcosip' ora' sp., Coll'totr'ichli1 sp., S.cp)to.
Rover had the least toxic effect fol- sp. and Stfempiliulini sp. were the me
lowed in ascending order of toxicity by common diseases found attacking chi
Bayleton, Saprol and Vondozeb. santhemum at different growth stagt
Rust and white rust caused by Pi'uri
In all the four fungicides, the safe spp. were the two other foliar disease
duration for feeding treated leaves to silk- attacking chrysanthemum in Baguio,
worms was 21 days after spraying. Trinidad, Benguet and Indang. Cavi
The yellow variety appears to be high
Diseases of Chrysanthemum in the Phil- susceptible to these diseases. Other d
ippines. G.G. Divinagracia,R.G. Bayot eases commonly observed were ste
and Olga N. Bayot, Dept. of Plant Pa- blight caused by Fusari imn sp. at
thology, University of the Philippines Erwinia icrysaifnI'mi and petal blig
at Los Bafios, College, Laguna. which appeared to be a complex disea
because several fungi such as Altcrin,
Surveys were conducted in commer- sp., Fusariim sp., Pnhtoplitlhor sp. at
cial chrysanthemum farms in Liliw, La- Stc'minlyliiiui sp. were found associall
guna; Ambiong, Baguio; in three with the disorder. A disease with viir
barangays in Cebu; Indang, Cavite; and like symptoms occu rred only in Bagui

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